Effect of dietary supplementation of broiler chickens with the natural antioxidants hesperidin and naringin on the expression of lipogenesis related genes and fatty acid profile

Hesperidin and naringin, flavonoids abundant in citrus fruits, exhibit health-promoting properties notably antioxidant and modulation of lipid metabolism. Increased antioxidant capacity and favorable fatty acid profile are desirable properties for broiler meat. In chickens hesperidin lowered plasma and egg yolk cholesterol and improved broiler meat antioxidant capacity. Here the effects of broiler diet supplementation with hesperidin and naringin on the expression of the lipogenesis related genes adiponectin, ppar-γ and fatty acid synthase (fasn) and fatty acid profile were assessed

Hesperidin and Naringin Improve Broiler Meat Fatty Acid Profile and Modulate the Expression of Genes Involved in Fatty Acid β-oxidation and Antioxidant Defense in a Dose Dependent Manner

The beneficial properties of the flavanones hesperidin and naringin as feed additives in poultry have lately been under investigation. In broilers, both flavanones have been shown to exhibit antioxidant properties while their individual effects on fatty acid (FA) composition and the underlying molecular mechanisms of their activity have not been explored. Here, we studied their effects on broiler meats’ FA profiles and on the expression of genes related to lipid metabolism, antioxidant defense and anti-inflammatory function. The experimental design comprised six treatment groups of broilers, each supplemented from day 11 until slaughter at 42 days with hesperidin, naringin or vitamin E, as follows: the E1 group received 0.75 g of hesperidin per kg of feed, E2 received 1.5 g hesperidin/kg feed, N1 received 0.75 g naringin/kg feed, N2 received 1.5 g naringin/kg feed, vitamin E (VE) received 0.2 g a-tocopheryl acetate/kg feed, and the control group was not provided with a supplemented feed. The VE treatment group served as a positive control for antioxidant activity. An analysis of the FA profiles of the abdominal adipose tissue (fat pad), major pectoralis (breast) and biceps femoris (thigh) muscles showed that both hesperidin and naringin had significant effects on saturated FA (SFA), polyunsaturated FA (PUFA) and omega n-6 content. Both compounds reduced SFA and increased PUFA and n-6 content, as well as reducing the atherogenicity and thrombogenicity indices in the breast muscle and fat pad. The effects on the thigh muscle were limited. An analysis of gene expression in the liver revealed that naringin significantly increased peroxisome proliferator-activated receptor alpha (PPARα), Acyl-CoA oxidase 1 (ACOX1) and glutathione disulfide reductase (GSR) expression. In the breast muscle, both hesperidin and naringin increased fatty acid synthase (FASN) expression and hesperidin increased the expression of adiponectin. In brief, both hesperidin and naringin supplementation beneficially affected FA profiles in the breast meat and fat pad of broiler chicken. These effects could be attributed to an increase in FA β-oxidation since the increased expression of related genes (PPARα and ACOX1) was observed in the liver. Furthermore, the antioxidant activity of hesperidin and naringin previously observed in the meat of broilers could be attributed, at least partly, to the regulation of antioxidant defense genes, as evidenced by the increased GSR expression in response to naringin supplementation

Characteristics of C57BL/6ByJ x 129P3/J F₂ mice fed low- and high-energy diets (Experiment 1).

<p>Values are mean±SD (minimum-maximum).</p><p>Age refers the age of the mouse at the end of the high-fat diet period.</p>*<p>Male versus female, p<0.05.</p

QTLs for percent body fat in C57BL/6ByJ×129P3/J F₂ mice (Experiment 1).

<p>Chr = chromosome. cM = centimorgan based on the experimental map. Marker = nearest LOD score peak. “Plus” refers to the allele that increases the trait value. Sex = sex-dependent by the criterion described in the text. For percent variance, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-t004" target="_blank"><b>Table 4</b></a>. For locus boundaries, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone-0068776-t005" target="_blank"><b>Table 5</b></a>.</p>a<p>Overdominance means that phenotype of heterozygotes differs from phenotypes of both homozygotes. *p<0.05. **p<0.01.</p

Comparison of QTLs from genome scans of B6 × 129 F₂ mice fed high-energy diets (Experiment 1) with published data.

<p>Chr = chromosome. C57BL/6J x 129S1/SvImJ refers to the results of a similar study that interbred these strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068776#pone.0068776-Su1" target="_blank">[36]</a>. C57BL/6ByJ x 129P3/J refers to the results reported here. If the locus boundaries overlap, the QTLs are considered the same. Other crosses that have overlapping obesity QTLs are shown with the strain conferring the allele that increases the trait value first. NA = not applicable.</p

Multiple regression analysis of variance for percent body fat in C57BL/6ByJ × 129P3/J F₂ mice fed low- and high-energy diets (Experiment 1).

<p><a href="mailto:Chr@Mb%E2%80%8A=%E2%80%8Achromosome/megabase" target="_blank">Chr@Mb = chromosome/megabase</a>. % Var = percent variance accounted for. Interactions are denoted by a semicolon. Degrees of freedom (df) are predicated on main effects and interactions.</p

Percent body fat of F₂ mice by sex and genotype (Experiment 1).

<p>Percent body fat of F₂ mice by sex and genotype (Experiment 1).</p

Percent body fat of F₂ mice by genotype of interacting loci detected in the pairwise genome scan (Experiment 1): chr 2 and 16 (upper left), chr 5 and 9 (upper right), and chr 3 and 13 (females, lower left; males, lower right).

<p>The x-axis shows genotype: BB, homozygous for C57BL/6ByJ alleles; PP, homozygous 129P3/J strain alleles; BP, heterozygous. Values are mean ± SEM. Groups that do not share a common subscript (a-f) significantly differed by post hoc testing.</p

QTL Analysis of Dietary Obesity in C57BL/6byj X 129P3/J F₂ Mice: Diet- and Sex-Dependent Effects

<div><p>Obesity is a heritable trait caused by complex interactions between genes and environment, including diet. Gene-by-diet interactions are difficult to study in humans because the human diet is hard to control. Here, we used mice to study dietary obesity genes, by four methods. First, we bred 213 F₂ mice from strains that are susceptible [C57BL/6ByJ (B6)] or resistant [129P3/J (129)] to dietary obesity. Percent body fat was assessed after mice ate low-energy diet and again after the same mice ate high-energy diet for 8 weeks. Linkage analyses identified QTLs associated with dietary obesity. Three methods were used to filter candidate genes within the QTL regions: (a) association mapping was conducted using >40 strains; (b) differential gene expression and (c) comparison of genomic DNA sequence, using two strains closely related to the progenitor strains from Experiment 1. The QTL effects depended on whether the mice were male or female or which diet they were recently fed. After feeding a low-energy diet, percent body fat was linked to chr 7 (LOD = 3.42). After feeding a high-energy diet, percent body fat was linked to chr 9 (<i>Obq5</i>; LOD = 3.88), chr 12 (<i>Obq34</i>; LOD = 3.88), and chr 17 (LOD = 4.56). The Chr 7 and 12 QTLs were sex dependent and all QTL were diet-dependent. The combination of filtering methods highlighted seven candidate genes within the QTL locus boundaries: <i>Crx</i>, <i>Dmpk</i>, <i>Ahr</i>, <i>Mrpl28</i>, <i>Glo1</i>, <i>Tubb5</i>, and <i>Mut</i>. However, these filtering methods have limitations so gene identification will require alternative strategies, such as the construction of congenics with very small donor regions.</p></div

Experimental design.

<p>In Experiment 1, B6 x 129 F₂ mice were fed a low- and then a high-fat diet. Body composition was measured at the end of each diet period, and QTL analyses were conducted for each diet condition. To narrow down a list of candidate genes that could account for the QTL, genotype association mapping (Experiment 2) was conducted using inbred strains and 4 million imputed SNPs. Differential gene expression analysis of tissues using microarrays (Experiment 3) indicated which genes in the QTL boundaries were differentially expressed between the parental B6 and 129 strains in liver, muscle, and adipose tissue. DNA genomic sequences from the QTL regions were compared between the parental strains (Experiment 4) to identify variants that might affect gene function. Results of all four experiments were compared to highlight genes identified by multiple methods.</p