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1.
J Dairy Sci ; 92(11): 5677-91, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19841227

ABSTRACT

Data from multiparous Holstein cows (n = 43) were used to determine whether supplementation of anions to low-potassium (K) prepartum diets would improve periparturient energy and macromineral status and affect performance during the postpartum period. Beginning 21 d before expected parturition, cows were fed a control diet (1.29% K; +10 mEq/100 g; n = 21) or a low dietary cation-anion difference (DCAD) diet (1.29% K; -15 mEq/100 g; n = 22) with anions provided through a combination of sulfate from calcium sulfate dihydrate (0.40% S total ration) and chloride (1.17% Cl total ration) from SoyChlor 16-7 (West Central, Ralston, IA). All cows were fed the same postpartum diet from parturition through 63 d postpartum. Feeding anions decreased overall urine pH (8.17 vs. 6.70) during the prepartum period. Overall, peripartum concentrations of plasma Ca, P, and Mg were similar between treatments; however, concentrations of plasma Ca tended to be increased during the first 24 h postcalving in cows fed the low DCAD diet. Overall, concentrations of plasma P tended to be increased by feeding the anionic diet prepartum; this effect was more pronounced during the immediate peripartal period. Anionic supplementation did not affect incidence of clinical (<5 mg/dL) and subclinical (5 to 8 mg/dL) hypocalcemia, clinical hypophosphatemia (<2 mg/dL), or clinical (<1.1 mg/dL) and subclinical (1.1 to 1.8 mg/dL) hypomagnesemia. Nevertheless, subclinical hypophosphatemia (2 to 4 mg/dL) tended to be decreased at 16 h postcalving and was decreased at d 2 postpartum for cows fed the anionic diet prepartum. Anion supplementation decreased prepartum dry matter intake (15.6 vs. 14.4 kg/d), but did not affect postpartum dry matter intake (22.4 vs. 23.0 kg/d), milk yield (46.5 vs. 46.1 kg/d), or content and yield of milk fat and true protein. Plasma concentrations of energy-related metabolites (glucose, nonesterified fatty acids, beta-hydroxybutyrate) were similar for both groups during the prepartum and postpartum periods. Glucose rate of appearance was determined by continuous infusion of 6,6-dideuterated glucose in a subset of cows between 6 and 10 d prepartum (control, n = 12; low DCAD, n = 9) and 7 and 10 d postpartum (control, n = 9; low DCAD, n = 8) periods. Glucose rate of appearance was not affected by treatment during the prepartum or postpartum periods. Overall, anion supplementation of low K diets improved P status during the early postpartum period, but did not affect aspects of energy metabolism or periparturient performance.


Subject(s)
Anions/administration & dosage , Cattle/physiology , Diet/veterinary , Dietary Supplements , Minerals/metabolism , Potassium, Dietary/metabolism , Animals , Body Constitution/physiology , Body Weight/physiology , Calcium/blood , Cattle/metabolism , Dairying , Eating/physiology , Energy Metabolism/physiology , Female , Hydrogen-Ion Concentration , Lactation , Least-Squares Analysis , Magnesium/blood , Milk/chemistry , Milk/metabolism , Minerals/blood , Phosphorus/blood , Postpartum Period , Pregnancy , Time Factors , Urine/chemistry
2.
J Dairy Sci ; 92(9): 4290-300, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19700689

ABSTRACT

Dietary lipid supplements have been extensively evaluated for their effects on mammary tissue mRNA abundance, including the classical lipogenic genes ACACA, SCD, FASN, and the transcription regulators SREBF1, THRSP, and PPARG. Novel gene isoforms with key regulatory roles in triacylglycerol synthesis have been recently identified including LPIN1 and AGPAT6. Transcriptional networks (i.e., genes whose mRNA expression is regulated by a transcription factor or nuclear receptor) coordinate adipogenesis and lipid filling in nonruminant adipose tissue. To investigate whether long-term milk fat depression affects adipogenic networks in subcutaneous adipose tissue, we characterized mRNA expression via quantitative PCR of 20 genes in cows fed saturated and polyunsaturated lipid for 3 wk. Adipose tissue from cows fed a control diet, control with fish (10 g/kg of dry matter) and soybean oil (25 g/kg of dry matter) (FSO), or control with saturated lipid (35 g/kg, EB100; Energy Booster 100, Milk Specialties, Dundee, IL) was biopsied after 21 d of feeding. Milk production did not differ across treatments (averaged 32 kg +/- 2.8 kg/d during the 21 d) but dry matter intake (DMI) decreased in cows fed FSO versus controls (averaged 18 vs. 22 kg/d during the 21 d). Despite the decrease in DMI, FSO resulted in similar energy intake as EB100 during the last 2 wk of the study. Cows fed FSO had a gradual decline in milk fat and energy yield leading to an overall 25% decrease in milk fat yield during the study (averaged 0.90 vs. 1.2 kg/d) compared with control or EB100. Thus, during the 21-d study, FSO led to a gradual increase in intake energy available for adipose tissue deposition. Relative mRNA expression of LPL and SCD as well as ADFP (coding for a protein involved in lipid droplet formation) and LPIN1 (coding for a protein involved in diacylglycerol synthesis/transcriptional regulation) was upregulated with FSO relative to other diets. Expression of the transcription regulator THRSP tended to be greater in cows fed FSO. Overall, results suggest that long-term milk fat depression caused by feeding FSO provided additional energy as well as long-chain fatty acids that, coupled with upregulation of a subset of adipogenic genes in subcutaneous adipose tissue, might have resulted in greater tissue lipid deposition.


Subject(s)
Cattle/physiology , Dietary Fats/metabolism , Dietary Supplements , Gene Regulatory Networks , Lactation/physiology , Milk/chemistry , Subcutaneous Fat/metabolism , Animals , Body Weight , Cattle/genetics , Cattle/metabolism , Diet/veterinary , Fats/metabolism , Female , Lipogenesis/genetics , Milk/metabolism , Time Factors
3.
J Dairy Sci ; 92(5): 2007-19, 2009 May.
Article in English | MEDLINE | ID: mdl-19389958

ABSTRACT

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.


Subject(s)
Dietary Supplements , Lactation , Lipids/administration & dosage , Mammary Glands, Animal/metabolism , Polymerase Chain Reaction/veterinary , Animals , Cattle , Female , Gene Expression Profiling , Genes/genetics , Lipogenesis/genetics , RNA, Messenger/metabolism
4.
J Dairy Sci ; 92(5): 2027-37, 2009 May.
Article in English | MEDLINE | ID: mdl-19389960

ABSTRACT

Studying long-chain fatty acid (LCFA) effects on gene network expression in bovine cells could provide useful information for future practical applications. An optimized in vitro system that does not require tissue collection or cell isolation could fill a niche in the study of PPARalpha activity in ruminants. Specific aims were to optimize culture conditions in Madin-Darby bovine kidney (MDBK) cells to achieve maximal mRNA expression of known peroxisome proliferator-activated receptor-alpha (PPARalpha) target genes using palmitate (16:0) as a representative LCFA. Variables included length of incubation time, use of albumin-bound (4:1 molar proportion) 16:0 (A16:0), or addition of insulin. A first time-course experiment tested culturing cells in Dulbecco's modified Eagle's medium with 150 microM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150 microM of 16:0 without albumin or insulin (-Alb/-Ins), 16:0 without albumin plus 5 mg/L of bovine insulin (-Alb/+Ins), A16:0 without insulin (+Alb/-Ins), or a control. A third experiment was a preliminary metabolic characterization of cells and assessed intracellular lipid droplet formation after treatment with 150 microM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24 h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARalpha target genes as well as PPARalpha coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (approximately 500%) and ACSL1 (50 to 200%) by 6 h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6 h only with A16:0. Further, there was a linear increase in expression of PPARA (approximately 100%) with A16:0 through 24 h of incubation. In experiment 2, insulin increased, and coupling LCFA with albumin tended to delay the response in expression of CPT1A and ACSL1 to 16:0. Data indicated a toxic effect of 150 microM free 16:0 as assessed by cell counts after 12 h of incubation. In experiment 3, MDBK cells appeared to use glucose and AA as energy sources and were able to secrete triglycerides. In addition, MDBK cells cultured with 150 microM of 16:0 had a substantial uptake of LCFA and synthesized intracellular lipid droplets. Overall, results indicated that a 6-h incubation with free LCFA and addition of insulin was suitable to detect marked effects on mRNA expression of PPARalpha target genes in MDBK cells.


Subject(s)
Fatty Acids/pharmacology , Gene Expression Regulation/drug effects , Genes/genetics , Kidney/cytology , Kidney/metabolism , PPAR alpha/metabolism , Palmitic Acid/metabolism , Animals , Cattle , Cell Culture Techniques , Cell Proliferation , Insulin/metabolism , Lipid Metabolism , Serum Albumin, Bovine/metabolism , Time Factors
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