condition: Control breed: German Landrace x German Edelschwein gender: male tissue: liver
Treatment protocol
The piglets were randomly allocated to two groups (group CON, group PIV) of n = 6 piglets each, and fed identical nutrient-adequate diets according to a phase feeding system (phase I: until a body weight of 15 kg; phase II: until the end of the experiment) throughout the experimental period of 28 days. The diet in phase I consisted of (g/kg): wheat, 381.7; barley, 315; soybean meal (44% crude protein), 250; soybean oil, 15; min-eral and vitamin premix, 33.5; L-lysin, 2.6; DL-methionine, 1.0 and L-threonine, 1.2. The diet of phase II consisted of (g/kg): wheat, 401.9; barley, 302; soybean meal (44% crude protein), 240; soybean oil, 15; mineral and vitamin premix, 33.4; L-lysin, 1.5; DL-methionine, 0.5 and L-threonine, 0.7 and a mixture of organic acids (containing 45% formic acid, 7.7% lactic acid, 5% sodium acetate), 5. The carnitine concentration in the diets was below 10 mg/kg diet as analyzed by LC-MS/MS. In ad-dition, piglets were given orally either (per kg body weight (BW)): NaHCO3 (Sig-ma-Aldrich, Steinheim, Germany) solution (60 mg/100 mL) (group CON) or 30 mg so-dium pivalate (95% purity; Sigma-Aldrich, Steinheim, Germany) dissolved in NaHCO3 solution (group PIV) each day throughout the experimental period of 28 days. Water was constantly available ad libitum from nipple drinkers during both experiments. At the end of the experiment, the pigs were killed in the post-prandial period (2 h after their last meal) by electronarcosis followed by exsanguination. Blood was col-lected into heparinized polyethylene tubes (Sarstedt, Nümbrecht, Germany) and plasma obtained by centrifugation at 1100 x g for 10 min at 4°C and subsequently stored at -80°C. Aliquots from liver and selected muscles (longissimus dorsi, semitendi-nosus, superficial biceps femoris) were collected, snap-frozen in liquid nitrogen and stored at -80°C pending analysis.
Growth protocol
The animal experiment was approved by the local Animal Care and Use Com-mittee (Regierungspräsidium Giessen; permission no: GI 19/3 No. 30/2013). All experimental procedures described followed established guidelines for the care and handling of laboratory animals. For the experiment a total of 12, 5-weeks-old, male cross-bred piglets (Pietrain x (German Landrace x German Edelschwein)) were used, which were kept in pairs in flat-deck pens under controlled conditions (23 ± 2°C room temperature, 50-60 % relative humidity, light from 06.00 a.m. to 07.00 p.m.).
Extracted molecule
total RNA
Extraction protocol
Total RNA from liver aliquots (20 mg) was extracted using TRIzol reagent (Invi-trogen, Karlsruhe, Germany) according to the manufacturer’s protocol. RNA quantity and quality were determined spectrophotometrically and by electrophoresis. For microarray hybridization, RNA samples were processed at the Center of Excellence for Fluorescent Bioanalytics (Regensburg, Germany).
Label
Biotin
Label protocol
200 ng of total RNA were used to generate double-stranded cDNA. 12 µg of subsequently synthesized cRNA was purified and reverse transcribed into sense-strand (ss) cDNA, whereat unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) followed by a terminal labeling with biotin.
Hybridization protocol
3.8 µg of fragmented and labeled ss cDNA were hybridized to Affymetrix Porcine Gene 1.0 ST arrays for 16 h at 45° C in a GeneChip hybridization oven 640.
Scan protocol
Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by the Affymetrix GeneChip Command Console v4.1.3 software.
Data processing
After scanning the arrays, cell intensity files containing a single intensity value for each probe cell were computed from the image data with the Affymetrix GeneChip Command Console Software. Summarized probe set signals in log2 scale were calculated by using the GCCN-SST-RMA algorithm with the Applied Biosystems GeneChip Expression Console v1.4 Software. After exporting into Microsoft Excel, average signal values, comparison fold changes (FC) and significance P-values were calculated.
