Dietary Inulin Supplementation Modifies Significantly the Liver Transcriptomic Profile of Broiler Chickens

<div><p>Inclusion of prebiotics in the diet is known to be advantageous, with positive influences both on health and growth. The current study investigated the differences in the hepatic transcriptome profiles between chickens supplemented with inulin (a storage carbohydrate found in many plants) and controls. Liver is a major metabolic organ and has been previously reported to be involved in the modification of the lipid metabolism in chickens fed with inulin. A nutrigenomic approach through the analysis of liver RNA hybridized to the Affymetrix GeneChip Chicken Genome Array identified 148 differentially expressed genes among both groups: 104 up-regulated (≥1.4-fold) and 44 down-regulated (≤0.6-fold). Quantitative real-time PCR analysis validated the microarray expression results for five out of seven genes tested. The functional annotation analyses revealed a number of genes, processes and pathways with putative involvement in chicken growth and performance, while reinforcing the immune status of animals, and fostering the production of long chain fatty acids in broilers supplemented with 5 g of inulin kg⁻¹ diet. As far as we are aware, this is the first report of a microarray based gene expression study on the effect of dietary inulin supplementation, supporting further research on the use of this prebiotic on chicken diets as a useful alternative to antibiotics for improving performance and general immunity in poultry farming, along with a healthier meat lipid profile.</p></div

ANOVA results of the genes studied in the liver by Real-time PCR assay from animals supplemented with 5 g of inulin kg⁻¹ diet and controls.

<p>ANOVA results of the genes studied in the liver by Real-time PCR assay from animals supplemented with 5 g of inulin kg⁻¹ diet and controls.</p

Stability of four reference genes on liver from animals supplemented with 5^-1 diet and controls, measured through three different software: Bestkeeper, GeNorm and NormFinder.

<p>Stability of four reference genes on liver from animals supplemented with 5^-1 diet and controls, measured through three different software: Bestkeeper, GeNorm and NormFinder.</p

Ingredients and nutrient composition of experimental control diet (g kg⁻¹ as fed basis).

1<p>Premix supplying (mg kg⁻¹ diet): 3 retinol, 55 cholecalciferol, 25 <i>dl-α-</i>tocopheryl acetate, 2.5 menadione, 3 thiamine, 6 riboflavin, 7 pyridoxine, 0.2 folic acid, 0.02 cyanocobalamin, 0.2 biotin, 25 calcium pantothenate, 50 niacin, 1300 choline chloride, 60 Mn, 80 Fe, 50 Zn, 5 Cu, 0.1 Se, 0.18 I, 0.5 Co, 0.5 Mo.</p>2<p>Determined.</p>3<p>Calculated.</p>4<p>SFA  =  saturated fatty acids; MUFA  =  monounsaturated fatty acids; PUFA  =  polyunsaturated fatty acids; UFA  =  unsaturated fatty acid.</p

List of chicken genes from KEGG pathway maps differentially expressed in liver from animals supplemented with 5⁻¹ diet and controls, with expression ratio, annotated gene description and KEGG ID.

<p>List of chicken genes from KEGG pathway maps differentially expressed in liver from animals supplemented with 5⁻¹ diet and controls, with expression ratio, annotated gene description and KEGG ID.</p

Differential expression results of the genes studied in the liver by Real-time PCR assay from animals supplemented with 5 g of inulin kg⁻¹ diet and controls using REST software (<i>P<0.08</i>).

<p>Differential expression results of the genes studied in the liver by Real-time PCR assay from animals supplemented with 5 g of inulin kg⁻¹ diet and controls using REST software (<i>P<0.08</i>).</p

Expression Levels of Genes Associated with the GH/IGF1 Axis, Oxidative Metabolism, and Antioxidant Defense in DEHP-Treated wt Mice

<p>Relative mRNA levels of genes involved in the GH/IGF1 growth axis, oxidative metabolism, and antioxidant defense in 13-wk-old wt mice treated with a low dose of the pro-oxidant DEHP. For each gene, expression levels in the treated wt mouse livers are plotted relative to that of age-matched untreated wt littermate controls (dotted line). Error bars indicate SEM. Asterisks indicate statistically significant differences (one-tailed <i>p</i> ≤ 0.05, see also Text S1).</p

Enhanced Sensitivity of <i>Csb^m/m/Xpa^−/−</i> Retinal Photoreceptor Cells to Genotoxic Insults

<p>Representative pictures of TUNEL stained retinas of 19-d-old wt, <i>Csb^m/</i>^m<i>, Xpa^−/−,</i> and <i>Csb^m/m/Xpa^−/−</i> mice (right panel), 20 h after exposure of animals to 10 Gy of ionizing radiation, and quantification of the number of TUNEL positive cells in the ONL (left panel). Note the significantly higher number of TUNEL-positive cells in the retina of <i>Csb^m/m/Xpa^−/−</i> mice, as compared to wt and single-mutant littermate controls (<i>p</i> < 0.05). Single asterisks indicate statistically significant differences between unirradiated <i>Csb^m/m/Xpa^−/−</i> and littermate control mice (<i>p</i> < 0.05), double asterisks indicate statistically significant differences between unirradiated and irradiated <i>Csb^m/m/Xpa^−/−</i> mice (<i>p</i> < 0.05).</p

Skeletal and Neurological Abnormalities in <i>Csb^m/m/Xpa^−/−</i> Mice

<div><p>(A) Radiographs of wt and <i>Csb^m/m/Xpa^−/−</i> mice (age as indicated) and photograph of a 15-d-old <i>Csb^m/m/Xpa^−/−</i> mouse (C57BL/6J).</p> <p>(B) 2-D images of micro-CT scans of tibiae taken from <i>Csb^m/m/Xpa^−/−</i> animals and wt littermate controls (age indicated in the figure). Horizontal sections are shown of the upper and lower part of the tibiae. e, epiphysis; g, growth plate; and d, diaphysis. I indicates the section through the smaller part of the diaphysis, II indicates section through the broader part of the diaphysis.</p> <p>(C) Growth of tibiae, taken from <i>Csb^m/m/Xpa^−/−</i> animals (light squares) and wt littermate controls (dark squares).</p> <p>(D) Representative footprint patterns of 19-d-old wt and <i>Csb^m/m/Xpa^−/−</i> mice. Arrows indicate the trajectory of each mouse. Stride length and front base width measurements on 15-d-old wt, <i>Xpa^−/−, Csb^m/m,</i> and <i>Csb^m/m/Xpa^−/−</i> mice. The significantly (asterisk; <i>p</i> < 0.001) greater base width in the double mutant mouse indicates ataxia.</p> <p>(E) Representative pictures of a TUNEL staining in the retina of wt and <i>Csb^m/m/Xpa^−/−</i> mice and quantification of the number of TUNEL positive cells. Arrows indicate TUNEL positive cells in the ONL and INL. Note the significantly higher number of TUNEL-positive cells in both the ONL and the INL in the retina of <i>Csb^m/m/Xpa^−/−</i> compared to wt mice (asterisk; <i>p</i> < 0.05).</p></div

Carbohydrate/Fat Metabolism and IGF1 Serum Levels

<p>IGF1 (A) and glucose (B) in the serum of 7-, 10-, 15-, and 17-d-old wt, <i>Xpa^−/−, Csb^m/m,</i> and <i>Csb^m/m/Xpa^−/−</i> mice (<i>n</i> = 6). The levels of IGF1 (ng/ml) and glucose (mmol/l) in the serum of <i>Csb^m/m/Xpa^−/−</i> mice are significantly lower than that of control littermates (<i>p</i> < 0.0004 and <i>p</i> < 0.04, respectively). (C) PAS staining for glycogen and Oil Red O staining for triglycerides in livers of 15-d-old wt and <i>Csb^m/m/Xpa^−/−</i> mice and 96-wk-old wt mice. Pictures were taken at 100× magnification. Note the large polyploid nuclei in the 96-wk-old wt mouse liver and the reduced glycogen levels in the <i>Csb^m/m/Xpa^−/−</i> liver after overnight fasting.</p