Three different diets differing in their main protein source but with comparable levels of gross energy and crude nutrients were fed. Nutrient concentrations in the diets were sufficient to meet the requirements according to German Society for Nutrition Physiology. In the diet of group CON, which contained soybean extraction meal as the main protein source, the amount of insect meal (IM) was 0 %, while in the diets of groups IM5 and IM10 the amount of IM was 5 % and 10 %, respectively, through partial (50 %) or complete (100 %) isonitrogenous replacement of SEM (44 % crude protein) by IM. The IM was produced from industrialized mass-rearing of Tenebrio molitor L.. The diets were offered ad libitum and feed consumption was recorded daily. BWs were recorded once per week. Water was constantly available ad libitum from a nipple drinker system. At day 29, the pigs were killed by electronarcosis followed by exsanguination. Aliquots of the liver were excised, washed in ice-cold NaCl solution (0.9 %) and snap-frozen in liquid nitrogen.
Growth protocol
The four-week feeding trial was approved by the local Animal Care and Use Committee (Regierungspräsidium Giessen; permission no: JLU 676_M). All experimental procedures described followed established guidelines for the care and handling of laboratory animals. The experiment included 30 five-week-old crossbreed pigs [Piétrain x (German Landrace x German Edelschwein)], which were randomly assigned to three groups of 10 pigs each [control (CON), 5 % insect meal (IM) (IM5), 10 % IM (IM10)], with similar initial body weights (BW) (CON: 8.66 ± 1.54 kg; IM5: 8.63 ± 1.49 kg; IM10: 8.79 ± 1.44 kg; mean ± SD; n = 10/group). The pigs were kept in groups of three or four animals in flat-deck pens under controlled conditions (23 ± 2°C room temperature, 50-60 % relative humidity, light from 07.00 a.m. to 07.00 p.m.).
Extracted molecule
total RNA
Extraction protocol
Total RNA from aliquots of liver (≈ 20 mg) was isolated using TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. RNA quantity and quality were assessed spectrophotometrically using an Infinite 200M microplate reader equipped with a NanoQuant plate (both from Tecan, Mainz, Germany). The average RNA concentration and the A260/A280 ratio of all total RNA samples (n = 30, means ± SD) were 2.06 ± 0.49 µg/µL and 1.97 ± 0.06, respectively. Transcript profiling in liver was carried out for 6 randomly selected pigs per group. Following an RNA quality check using an Agilent 2100 Bioanalyzer (Agilent technologies, Böblingen, Germany), the total RNA samples were processed at the Kompetenzzentrum Fluoreszente Bioanalytik (Regensburg, Germany) using an Affymetrix GeneChip Porcine Gene 1.0 Sense Target array, which represents 19,212 porcine genes, according to the manufacturer´s instructions. The average RNA integrity number (RIN) values of all samples (n = 18, means ± SD) were 7.91 ± 0.28 (liver).
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.
