Transcriptomic profiling of jejunum mucosa of two laying hen strains in response to low dietary Phosphorus
Transcriptomic profiling of jejunum mucosa of two laying hen strains in response to low dietary Phosphorus
Forty LB and 40 LSL hatchlings were obtained from Lohmann Tierzucht GmbH, Cuxhaven, Germany. Hens were housed individually in metabolism crates starting at 15 or 20 weeks of age. The experimental diets were provided for four weeks before hens were slaughtered at weeks 19 and 24. This experiment had a 2×2×2-factorial arrangement of treatments with hen strain, hen age (19 and 24 weeks, representing before and after the onset of laying), and mineral P supplement (0 and 1 g P/kg of feed). Diets were maize-soybean meal-based, without a phytase supplement, and otherwise composed to contain all nutrients at recommended levels. The P concentration in the plant-based diet, without a mineral P supplement, was about 3 g/kg, equivalent to approximately 1.5 g/kg of NPP. Ten hens per strain and age were sampled, according to the diet treatments (80 hens in total), and each pen served as one experimental unit for all measurements in the statistical analysis. The hens were killed by electronic stunning and exsanguination from 07:00 to 13:00. Jejunum samples approximately 2 cm were collected from approximately 3 cm distal to the duodenal loop. The samples were cut open; the mucosa was thoroughly rinsed with a 0.9% NaCl solution, and scraped for each bird. The small intestinal mucosa (jejunum) samples were immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Approximately 50 mg of jejunal mucosal tissues were used for RNA isolation using TRI reagent according to the manufacturer's protocol (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). DNase treatment was performed to remove DNA fragments. RNA quality and quantity were determined using NanoDrop ND-2000 (Peqlab, Erlangen, Germany), a Qubit Fluorometer (Thermo Fisher Scientific, Germany) and a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany). High quality RNA (RNA integrity number; RIN > 7.5)  Total RNA of one microgram (RIN > 7.5) was used for library preparation using an Illumina Stranded mRNA Prep, Ligation kit with 10 PCR-cycles of amplification according to the manufacture's recommendation (Illumina). The libraries were quality checked for fragment length distribution on Bionalyzer and normalized to an equal concentration of 10nM prior to pooling.