Dietary bile acids supplementation improves the growth performance and alleviates fatty liver in broilers fed a high-fat diet via improving the gut microbiota.

This experiment aims to evaluate the effect of bile acids (BAs) in alleviating fatty liver disease induced by a high-fat diet (HFD) in broilers, and the modulation of the gut microbiota involved in this process. A total of 192 one-day-old Arbor Acres (AA) commercial male broilers were randomly divided into 4 groups and treated with the following diet: a basal-fat diet (BFD), a basal-fat diet plus bile acids (BFD + BAs), an HFD, and a high-fat diet plus bile acids (HFD + BAs). Bile acids were supplemented at the early growth stage (3-7 d), middle stage (17-21 d), and late stage (31-35 d). Results showed that BAs treatment had a significant effect on body weight on 14 d and 35 d, and increased the breast muscle weight and its index, but decreased the liver weight and abdominal fat weight on 35 d (P < 0.05). The supplementation of BAs significantly improved the serum lipid profile and decreased the level of triglycerides (TG), total cholesterol (TCHO), and nonesterified fatty acids (NEFA) on 35 d (P < 0.05). Dietary BAs supplementation significantly alleviated the hepatic TG deposition induced by HFD (P < 0.05), which was accompanied by upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) gene expression (P < 0.05). Moreover, the expression levels of hepatic gene adipose triglyceride lipase (ATGL), peroxisome proliferator-activated receptor α (PPARα), and apolipoprotein B (APOB) were greatly increased by BAs treatment. The analysis of 16S rRNA sequencing showed that the microbial diversity of the cecal digesta was increased by BAs in broilers with elevated abundances of Firmicutes, Lactobacillus, Anaerostipes, Sellimonas, and CHKCI002 and decreased abundances of Barnesiella and Akkermansia genus (P < 0.05). Hepatic TG content was positively correlated with the abundance of Oscillospiraceae, but it was negatively correlated with the abundance of Lactobacillus in cecal digesta (P < 0.05). These results indicate that dietary BAs can improve growth performance and alleviate fatty liver disease induced by an HFD via modulating gut microbiota in broilers.

α-Lipoic Acid Alleviates Hepatic Lipid Deposition by Inhibiting FASN Expression via miR-3548 in Rats.

Excessive liver lipid deposition is a vital risk factor for the development of many diseases. Here, we fed Sprague-Dawley rats with a control or α-lipoic acid-supplemented diet (0.2%) for 5 weeks to elucidate the effects of α-lipoic acid on preventive ability, hepatic lipid metabolism-related gene expression, and the involved regulatory mechanisms. In the current study, α-lipoic acid supplementation lowered plasma triglyceride level and hepatic triglyceride content. Reduced hepatic lipid deposition was closely associated with inhibiting fatty acid-binding protein 1 and fatty acid synthase expression, as well as increasing phosphorylated hormone-sensitive lipase expression at the protein level in α-lipoic acid-exposed rats. Hepatic miRNA sequencing revealed increased expression of miR-3548 targeting the 3'untranslated region of Fasn mRNA, and the direct regulatory link between miRNA-3548 and FASN was verified by dual-luciferase reporter assay. Taken together, α-lipoic acid lowered hepatic lipid accumulation, which involved changes in miRNA-mediated lipogenic genes.

Dietary betaine supplementation in hens modulates hypothalamic expression of cholesterol metabolic genes in F1 cockerels through modification of DNA methylation.

Betaine is widely used in animal nutrition to promote growth, development and methyl donor during methionine metabolism through nutritional reprogramming via regulation of gene expression. Prenatal betaine exposure is reported to modulate hypothalamic cholesterol metabolism in chickens, yet it remains unknown whether feeding hens with betaine-supplemented diet may affect hypothalamic cholesterol metabolism in F1 offspring. In this study, hens were fed with basal or betaine-supplemented (0.5%) for 30days, and the eggs were collected for incubation. The hatchlings were raised under the same condition up to 56days of age. Betaine-treated group showed significantly (P<0.05) higher plasma concentration of total cholesterol and HDL-cholesterol, together with increased hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic gene expression of SREBP2, HMGCR, and LDLR was significantly up regulated (P<0.05). Also, mRNA abundances of SREBP1, ACAT1 and APO-A1 were up-regulated, while that of CYP46A1 was significantly down-regulated (P<0.05). These changes coincided with a significant down-regulation of BDNF and CRH, and a significant up-regulation of NPY mRNA expression. Moreover, genes involved in methyl transfer cycle were also modulated. DNMT1 and BHMT were up-regulated (P<0.05) at both mRNA and protein levels, which was associated with significant modifications of CpG methylation on the promoter of SREBP-1, SREBP-2 and APO-A1 genes as detected by bisulfate sequencing. These results indicate that feeding betaine to hens modulates hypothalamic expression of genes involved in cholesterol metabolism and brain functions in F1 cockerels with modification of promoter DNA methylation.

Embryonic exposure to corticosterone modifies aggressive behavior through alterations of the hypothalamic pituitary adrenal axis and the serotonergic system in the chicken.

Exposure to excess glucocorticoids (GCs) during embryonic development influences offspring phenotypes and behaviors and induces epigenetic modifications of the genes in the hypothalamic-pituitary-adrenal (HPA) axis and in the serotonergic system in mammals. Whether prenatal corticosterone (CORT) exposure causes similar effects in avian species is less clear. In this study, we injected low (0.2µg) and high (1µg) doses of CORT into developing embryos on day 11 of incubation (E11) and tested the changes in aggressive behavior and hypothalamic gene expression on posthatch chickens of different ages. In ovo administration of high dose CORT significantly suppressed the growth rate from 3weeks of age and increased the frequency of aggressive behaviors, and the dosage was associated with elevated plasma CORT concentrations and significantly downregulated hypothalamic expression of arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH). The hypothalamic content of glucocorticoid receptor (GR) protein was significantly decreased in the high dose group (p<0.05), whereas no changes were observed for GR mRNA. High dose CORT exposure significantly increased platelet serotonin (5-HT) uptake, decreased whole blood 5-HT concentration (p<0.05), downregulated hypothalamic tryptophan hydroxylase 1 (TPH1) mRNA and upregulated 5-HT receptor 1A (5-HTR1A) and monoamine oxidase A (MAO-A) mRNA, but not monoamine oxidase B (MAO-B). High dose CORT also significantly increased DNA methylation of the hypothalamic GR and CRH gene promoters (p<0.05). Our findings suggest that embryonic exposure to CORT programs aggressive behavior in the chicken through alterations of the HPA axis and the serotonergic system, which may involve modifications in DNA methylation.

LPS-induced inflammation in the chicken is associated with CCAAT/enhancer binding protein beta-mediated fat mass and obesity associated gene down-regulation in the liver but not hypothalamus.

BACKGROUND: The fat mass and obesity associated gene (FTO) is widely investigated in humans regarding its important roles in obesity and type 2 diabetes. Studies in mammals demonstrate that FTO is also associated with inflammation markers. However, the association of FTO with inflammation in chickens remains unclear. In this study, male chickens on day 28 posthatching were injected intraperitoneally with lipopolysaccharide (LPS) or saline to investigate whether the FTO gene is involved in LPS-induced inflammation. RESULTS: We detected significant down-regulation of FTO mRNA in the liver (P < 0.01), but not in the hypothalamus, 2 and 24 h after LPS challenge. Toll-like receptor (TLR) 2 (P < 0.01) and TLR4 (P < 0.01) followed the same pattern as FTO, being suppressed significantly in liver but not in hypothalamus. IL-1ß was dramatically up-regulated (P < 0.01) in both liver and hypothalamus 2 h after LPS challenge, while activation of IL-6 was observed in the liver (P < 0.01), but not in hypothalamus. The 5'-flanking sequence of the chicken FTO gene contains nine predicted binding sites for CCAAT/enhancer binding protein beta (C/EBP beta) and one for signal transducer and activator of transcription 3 (STAT3). Significant elevation of C/EBP beta was detected in the liver (P < 0.01), but not in the hypothalamus, 2 h after LPS challenge. Lipopolysaccharide challenge increased the C/EBP beta binding to FTO promoter in the liver (P < 0.01 for fragment 1, P < 0.05 for fragment 2), although the protein content of C/EBP beta was not altered. Moreover, injection of LPS resulted in enhanced phosphorylation of liver STAT3, a downstream transcription factor in IL-6 signaling. Although phosphorylated STAT3 was not detected to directly bind to FTO promoter, it was found to interact with C/EBP beta. CONCLUSION: Our results reveal that FTO expression in liver, but not in hypothalamus, is affected by the i.p. injection of LPS, which may be mediated through tissue-specific FTO transcriptional regulation by C/EBP beta and STAT3 interaction.

Antioxidant effect of a phytoestrogen equol on cultured muscle cells of embryonic broilers.

Previous studies have shown that the in ovo injection of equol can markedly improve the water-holding capacity of muscles of broilers chickens at 7 wk of age through promotion of the antioxidant status. We aimed to investigate directly the antioxidant effects of equol on muscle cells in broilers. Muscle cells were separated from leg muscle of embryos on the 11th day of incubation and treated with equol and H(2)O(2), either alone or together. Cells were pretreated with medium containing 1, 10, or 100 µM equol for 1 h prior to the addition of 1 mM H(2)O(2) for a further 1 h. Photomicrographs of cells were obtained. Cell viability, malondialdehyde (MDA) content, and L-lactate dehydrogenase (LDH) activity in the cell supernatant, as well as intracellular total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activities were determined. Treatment with 1 mM H(2)O(2) caused serious damage to cells, indicated by comets with no clear head region but a very apparent tail of DNA fragments. Pretreatment with low (1 µM) but not high concentrations of equol (10 µM) inhibited cell damage, while 100 µM equol caused more serious damage than H(2)O(2) alone. Pretreatment with 1 µM equol had no effect on cell viability, while pretreatment with 10 and 100 µM equol significantly decreased cell viability in a dose-dependent manner. Compared with H(2)O(2) alone, pretreatment with low-dosage equol markedly decreased LDH activity and MDA production in the supernatant, significantly increased intracellular T-SOD activity (P < 0.05) and tended to increase intracellular GSH-Px activity (0.05 < P < 0.1). Pretreatment with high-dosage equol (10 and 100 µM) significantly enhanced LDH activity, but had no effect on MDA content, T-SOD or GSH-Px activity induced by H(2)O(2,) except for an obvious increase in GSH-Px activity caused by 10 µM equol. These results indicate that equol at low dosage can prevent skeletal muscle cell damage induced by H(2)O(2), while pretreatment with high-dosage equol shows a synergistic effect with H(2)O(2) in inducing cell damage.

Dual effects of daidzein on chicken hepatic vitellogenin II expression and estrogen receptor-mediated transactivation in vitro.

Two in vitro systems were employed to delineate the estrogenic activity of daidzein (Da), alone or in combination with high or low concentrations of estrogen in two cell types possessing different estrogen-receptor (ER) isoforms, ERalpha and/or ERbeta: (1) vitellogenin II (VTG), the egg yolk precursor protein and the endpoint biomarker for estrogenicity, in chicken primary hepatocytes, and (2) CHO-K1 cells transiently co-transfected with ERalpha or ERbeta and estrogen-response elements (ERE) linked to a luciferase reporter gene. Da (100 microM) alone induced VTG mRNA expression in chicken hepatocytes, albeit with much less potency compared to estradiol (E(2)). Da exhibited different effects in the presence of 1 microM and 10 microM E(2). At a concentration of 100 microM, Da enhanced 1 microM E(2)-induced VTG transcription by 2.4-fold, but significantly inhibited 10 microM E(2)-induced VTG mRNA expression in a dose-dependent fashion from 1 to 100 microM. Tamoxifen completely blocked the estrogenic effect of daidzein, alone or in combination with 1 microM of E(2), but did not influence its anti-estrogenic effect on 10 microM E(2)-induced VTG mRNA expression. Furthermore, neither E(2) nor daidzein, alone or in combination, affected ERalpha mRNA expression, yet all the treatments significantly up-regulated ERbeta mRNA expression in chicken hepatocytes. E(2) effectively triggered estrogen-response elements (ERE)-driven reporter gene transactivation in CHO-K1 cells expressing ERalpha or ERbeta and showed much greater potency with ERalpha than with ERbeta. In contrast, daidzein was 1000 times more powerful in stimulating ERbeta- over ERalpha-mediated transactivation. Daidzein, in concentrations ranging from 5 nM to 50 microM, did not affect ERbeta-mediated transactivation induced by 1 nM E(2), but it significantly inhibited ERbeta-mediated transactivation induced by 10 nM E(2) at 500 nM. Despite the tremendous difference in sensitivity between the two in vitro systems, daidzein exhibited greater potency as an estrogen-antagonist for ERbeta-mediated activity.

Layer and broiler chicks exhibit similar hypothalamic expression of orexigenic neuropeptides but distinct expression of genes related to energy homeostasis and obesity.

Layer and broiler chickens demonstrate striking differences in body weight and body composition. However, the mechanism underlying such difference is elusive. Hypothalamus-pituitary-adrenal (HPA) axis regulates energy homeostasis and body size in mammals, but information in birds is scarce. Here we test the hypothesis that such breed difference is more associated with hypothalamic expression of genes related to HPA axis, rather than orexigenic neuropeptides. Broiler chicks exhibit significantly higher body weight and food intake at day (D) 7 posthatching, but the food intake relative to body weight gain was actually lower. No breed differences were observed for hypothalamic expression of neuropeptide Y (NPY), agouti-related protein (AGRP), proopiomelanocortin (POMC), orexin (ORX), leptin receptor (LEPR), acetyl-CoA carboxylase (ACC) or fatty acid synthase (FAS). However, broiler chicks expressed significantly higher glucocorticoid receptor (GR) mRNA (P<0.05) and protein (P<0.01) in hypothalamus compared to layer chicks, which is associated with lower corticotropin-releasing hormone (CRH) mRNA (P<0.05) yet higher accumulation of CRH peptide in hypothalamus, suggesting an augmented GR-mediated negative feedback regulation of CRH transcription and release in broiler chicks. Furthermore, fat mass and obesity associated (FTO) gene was also more highly expressed in hypothalamus of broiler chicks (P<0.05). These results suggest that the genes related to energy homeostasis and obesity, such as GR, CRH and FTO, rather than orexigenic neuropeptides, are impacted by the genetic selection practices and play a role in breed-specific body weight setpoint regulation in the chicken.

Effect of dietary daidzein on egg production, shell quality, and gene expression of ER-alpha, GH-R, and IGF-IR in shell glands of laying hens.

Our previous studies demonstrated that dietary daidzein improves egg production in ducks during the late period of the laying cycle. The present study was aimed to investigate the effect of daidzein in laying hens, with more focus on eggshell quality. The expression of ER-alpha, GH-R, and IGF-IR mRNA in shell glands was determined to identify the target genes of daidzein action and to reveal the relationship between shell quality and profiles of gene expression in shell glands of laying hens. 1000 ISA hens, at 445 days of age, were allotted at random to two groups and given the basal diet with or without 10 mg of daidzein per kg diet for 9 weeks. Daidzein supplement significantly increased the egg laying rate and the feed conversion ratio. The eggshell thickness increased, while the percentage of cracked eggs decreased in daidzein-treated hens. Serum E2 and phosphate concentrations were not altered, but the level of serum Ca2+ and the tibia bone mineral density were significantly increased in the daidzein-treated group compared with their control counterparts. In parallel with the significant increase of oviduct weight, significant down-regulation of GH-R and IGF-IR mRNA and a trend of decrease in ERalpha mRNA expression in shell glands were observed in daidzein-treated hens. The results indicate that dietary daidzein improves egg laying performance and eggshell quality during the late (postpeak) laying stage of hens, which is associated with modulations in gene expression in the shell gland.

Dietary daidzein influences laying performance of ducks (Anas platyrhynchos) and early post-hatch growth of their hatchlings by modulating gene expression.

Our previous studies demonstrated that dietary supplementation of daidzein improves egg production in duck breeders during late periods of the laying cycle. The present study was aimed to clarify whether the growth of ducklings hatched from eggs laid by daidzein-treated hens would be affected, and to elucidate the mechanisms underlying potential trans-generational effects, by determining changes of hormone levels and mRNA expression of relevant genes. Daidzein was added to the basal diet of 415-day-old duck breeders at the level of 5 mg/kg. During 9 weeks of daidzein treatment, laying rate increased by 7.70%, average egg mass tended to increase, whereas yolk/albumen ratio decreased significantly. These changes were accompanied by significantly elevated plasma T4 and E2 levels, enhanced gonadotropin releasing hormone (GnRH) mRNA, but diminished estrogen receptor (ER)-beta mRNA expression in hypothalamus of daidzein-treated hens. Ducklings hatched from daidzein-treated eggs were significantly smaller at hatching, but they caught up with their control counterparts by 4 weeks of age. Serum levels of T4, pituitary GH, hepatic GH receptor (GHR) and type-1 IGF receptor (IGF-1R) mRNA expression were all suppressed markedly in the daidzein-treated group at hatching, but this suppression proved to be temporary, as at 4 weeks of age, expression levels of all investigated genes were restored. However, it is noteworthy that at 4 weeks of age an obvious down-regulation of hypothalamic GnRH mRNA expression was detected in ducklings maternally exposed to daidzein. Our results provide evidence that maternal exposure to daidzein affects post-hatch growth in the duck with accompanying changes in the secretion of metabolic hormones and expression of growth-related genes. Although the negative effect of maternal daidzein on embryonic growth could be eliminated 4 weeks after hatching, the long-term effect of maternal daidzein on reproductive function is not to be ignored and awaits further investigation.