Effects of alfalfa saponin extract on the performance and cholesterol metabolism of 1 laying hens 2

Abstract 31 The experiment was performed to determine the effects of alfalfa saponin extract (ASE

Digital Gene-Expression Profiling Analysis of the Cholesterol-Lowering Effects of Alfalfa Saponin Extract on Laying Hens

<div><p>Background</p><p>To prevent cardiovascular disease, people are advised to limit their intake of dietary cholesterol to less than 300 mg/day. Egg consumption has been seriously reduced because of the high levels of cholesterol. The purpose of the present study was to evaluate the cholesterol-lowering effects of alfalfa saponin extract (ASE) in yolk and the molecular mechanisms underlying these effects using digital gene-expression profiling analysis. Liver and ovary tissues were isolated from laying hens fed with ASE for RNA sequencing.</p><p>Results</p><p>The cholesterol content of the yolks of eggs from hens fed 120 mg/kg ASE declined considerably on day 60. Other groups (60, 240, 480 mg/kg ASE group) also showed decreases, but they were not significant. Digital gene expression generated over nine million reads per sample, producing expression data for least 12,384 genes. Among these genes, 110 genes showed greater than normal expression in the liver and 107 genes showed greater than normal expression in the ovary. Cholesterol 7 alpha-hydroxylase (<i>Cyp7a1</i>) and apolipoprotein H (<i>Apoh</i>), which act in the synthesis of bile acid and cholesterol efflux, showed more expression in the livers of hens given dietary ASE supplementation. In the ovary, levels of very low density lipoprotein receptor (<i>Vldlr</i>), apolipoprotein B (<i>Apob</i>), apovitellenin 1 (<i>ApovldlII</i>) and vitellogenin (<i>VtgI</i>, <i>VtgII</i> and <i>VtgIII</i>) in ovary decreased with dietary ASE supplementation.</p><p>Conclusion</p><p>Transcriptome analysis revealed that the molecular mechanisms underlying the cholesterol-lowering effects of ASE were partially mediated by enhancement of cholesterol efflux in the liver and this reduced of cholesterol deposition in the ovary.</p></div

GO functional enrichment of genes differentially expressed in the liver and ovary.

<p>Gene classification based on gene ontology (GO) for differentially expressed genes in the liver and ovaries of laying hens. The number of genes in GO terms was analyzed using GO Slim Assignment. The <i>y-axis</i> and <i>x-axis</i> indicate the number of genes in each cluster and the names of clusters, respectively. Only biological processes were used for GO analysis.</p

Composition and nutrient content of diet.

<p>Note: ¹Premix provided the following per kilogram of diet: 60 mg of iron, 80 mg of manganese, 8 mg of copper, 80 mg of zinc, 1 mg of iodine, 0.3 mg of selenium, 12,200 IU of vitamin A, 4125 IU of vitamin D₃, 30 IU of vitamin E, 4.5 mg of vitamin K, 5 mg of vitamin B₁₂, 2 mg of biotin, 5 mg of folic acid, 32.5 mg of niacin, 5.3 mg of pantothenic acid, 8 mg of pyridoxine, 8.5 mg of riboflavin, and 1 mg of thiamin.</p>2<p>Obtained by calculation.</p

Saturation curve analysis of the digital gene expression tag libraries generated from four samples.

<p>A–D denote Li_CK, Li_Exp, Ov_CK and Ov_Exp, respectively. The relative number of genes identified (y axis) increased as the total tag number of tags (x axis) increased.</p

Dietary ASE and concentrations of yolk cholesterol (mg/g).

a,b<p>Means in the same row not sharing a common superscript differ significantly at <i>P</i><0.05.</p

ASE and laying performance.

a,b<p>Means in the same row not sharing a common superscript differ significantly at <i>P</i><0.05.</p

Sequencing and qRT-PCR.

<p>Quantitative RT-PCR validation of differentially expressed genes in control (a basal diet) and treatment (diet with 120 mg/kg ASE) samples from liver and ovary tissues, including eight genes. All data were normalized to the expression level of actin. Data represent fold change of relative quantification in treatment vs. control samples. The error bars represent the range of the fold change as determined by the data assist software.</p

Basic characteristics of tags in four libraries and data of sequencing reads mapping to the reference genome.

<p>Note: 1. “+” refers to sense strands, “−” refers to anti-sense strands.</p><p>2. “Non-splice reads” refers to reads for the entire sequence is mapped to one exon; “Splice reads” also called junction reads, refers to reads mapped to the border of exon.</p