gender: female (cow) condition: control tissue: liver breed: Holstein
Treatment protocol
the cows received either corn and grass silage-based antepartum (a.p.) or p.p. total mixed rations (control group, n = 11) or the same diets supplemented with rumen-protected NA (NiaShure; Balchem, New Hampton, NY, USA) (NA group, n = 11) at a dosage of 79 mg/kg BW. The diets were calculated to meet energy and protein requirements of dairy cows following the recommendations of the German Society of Nutrition Physiology (GfE, 2001). Considering the rumen degradability of NiaShure (Zeitz et al., 2018), the applied NA dosage was equivalent to 47 mg NA at the duodenum and estimated to be equivalent to 31 mg of absorbable NA per kg BW based on information given by the supplier. This NA dosage was close to that required for pharmacological use as an antilipidaemic drug in humans (approximately 10-30 mg NA/kg BW) (Offermanns et al., 2006). Biopsies of the liver were taken 1 wk after calving, and the liver tissue was snap-frozen in liquid N2 and stored at −80°C. More details about sample collection are given by Zeitz et al. (2018).
Growth protocol
A total of 22 multiparous Holstein dairy cows were used which were part of a study carried out at the Educational and Research Centre for Animal Husbandry, Hofgut Neumühle (Münchweiler an der Alsenz, Germany) and approved by the Provincial Government of Coblenz, Germany (23 177-07/G 14-20-050). The experimental period lasted from 3 wk before expected calving until 1 wk p.p. Details on feeding and husbandry conditions are given in Zeitz et al. (2018).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from liver samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and stored at -80 °C. Concentration and purity of the RNA were estimated from the optical density at 260 and 280 nm, respectively, using a NanoQuant plate (Infinite 200, Tecan, Mainz, Germany). Prior to sample processing at the Centre of Excellence for Fluorescent Bioanalytics at the University of Regensburg, the concentration and integrity of RNA was analyzed using an Agilent 2100 Bioanalyzer (Agilent technologies, Böblingen, Germany). The total RNA concentrations, optical density A260/A280 ratios and RNA integrity number (RIN) values of all samples were 0.22 ± 0.08 µg/µL, 2.00 ± 0.03 and 7.2 ± 0.5 (mean ± SD, n = 18), respectively. From the 11 available RNA samples per group, the two samples with the lowest RIN values were excluded.
Label
Biotin
Label protocol
The cRNA was labeled with biotin using the Affymetrix GeneChip labeling kit.
Hybridization protocol
After checking the quality and quantity of the labeled cRNA, cRNA was fractionated and hybridized with the Affymetrix GeneChips. GeneChips were washed and stained with the Affymetrix GeneChip Fluidics station 450. The GeneChips were then scanned with an Affymetrix GeneChip scanner 3000. All procedures were performed according to Affymetrix protocols (GeneChip expression analysis, Technical manual from Affymetrix). The quality of hybridization was assessed in all samples following the manufacturer´s recommendations.
Scan protocol
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.
Data processing
Probe cell intensity data were further analyzed in the Affymetrix Expression Console software using the Robust Multichip Analysis (RMA) algorithm.
