Curcumin alleviates cecal oxidative injury in diquat-induced broilers by regulating the Nrf2/ARE pathway and microflora.

This study evaluated the alleviative effect of curcumin (CUR) on the diquat (DQ)-induced cecal injury in broilers. A total of 320 one-day-old Cobb broilers were selected and randomly divided into 4 treatments, namely control, DQ, CUR 100, and CUR150 groups. The control and DQ groups were fed a basal diet, while the CUR 100 and CUR150 groups were fed the basal diet supplemented with 100 and 150 mg/kg CUR, respectively. Each group had 8 replicates, with 10 broilers per replicate. On day 21 of the experiment, 1 broiler was selected from each replicate and intraperitoneally injected 20 mg/kg body weight of DQ for DQ, CUR 100, and CUR 150 groups. Broilers in control group received equivalent volume of saline. Broilers were euthanized 48h postinjection for tissue sampling. The results showed that DQ injection could cause oxidative stress and inflammatory reactions in the cecum, affecting the fatty acid production and flora structure, thus leading to cecum damage. Compared with the DQ group, the activity of superoxide dismutase, the level of interleukin 10, acetic acid, and total volatile fatty, and the abundance of nuclear factor E2-related factor 2, copper and zinc superoxide dismutase and catalase mRNA in the cecal mucosa of broilers in the CUR group increased significantly (P < 0.05). However, the levels of malondialdehyd, reactive oxygen species, tumor necrosis factor-alpha, and the expression of cysteine-aspartic acid protease-3 and tumor necrosis factor-alpha decreased significantly (P < 0.05) in the CUR group. In addition, CUR treatment alleviated the damage to the cecum and restored the flora structure, and Lactobacillus and Lactobacillaceae promoted the alleviative effect of CUR on DQ. In summary, CUR could alleviate the cecal injury caused by DQ-induced oxidative damage and inflammatory reactions by regulating the Nrf2-ARE signaling pathway and intestinal flora, thus protecting the cecum.

Transcriptomic Analysis of Laying Hens Revealed the Role of Aging-Related Genes during Forced Molting.

Molting in birds provides us with an ideal genetic model for understanding aging and rejuvenation since birds present younger characteristics for reproduction and appearance after molting. Forced molting (FM) by fasting in chickens causes aging of their reproductive system and then promotes cell redevelopment by providing water and feed again. To reveal the genetic mechanism of rejuvenation, we detected blood hormone indexes and gene expression levels in the hypothalamus and ovary of hens from five different periods during FM. Three hormones were identified as participating in FM. Furthermore, the variation trends of gene expression levels in the hypothalamus and ovary at five different stages were found to be basically similar using transcriptome analysis. Among them, 45 genes were found to regulate cell aging during fasting stress and 12 genes were found to promote cell development during the recovery period in the hypothalamus. In addition, five hub genes (INO80D, HELZ, AGO4, ROCK2, and RFX7) were identified by WGCNA. FM can restart the reproductive function of aged hens by regulating expression levels of genes associated with aging and development. Our study not only enriches the theoretical basis of FM but also provides insights for the study of antiaging in humans and the conception mechanism in elderly women.

Optimization of whole-brain rabies virus tracing technology for small cell populations.

The lateral hypothalamus (LH) is critically involved in the regulation of homeostatic energy balance. Some neurons in the LH express receptors for leptin (LepRb), a hormone known to increase energy expenditure and decrease energy intake. However, the neuroanatomical inputs to LepRb-expressing LH neurons remain unknown. We used rabies virus tracing technology to map these inputs, but encountered non-specific tracing. To optimize this technology for a minor cell population (LepRb is not ubiquitously expressed in LH), we used LepRb-Cre mice and assessed how different titers of the avian tumor virus receptor A (TVA) helper virus affected rabies tracing efficiency and specificity. We found that rabies expression is dependent on TVA receptor expression, and that leakiness of TVA receptors is dependent on the titer of TVA virus used. We concluded that a titer of 1.0-3.0 × 107 genomic copies per µl of the TVA virus is optimal for rabies tracing. Next, we successfully applied modified rabies virus tracing technology to map inputs to LepRb-expressing LH neurons. We discovered that other neurons in the LH itself, the periventricular hypothalamic nucleus (Pe), the posterior hypothalamic nucleus (PH), the bed nucleus of the stria terminalis (BNST), and the paraventricular hypothalamic nucleus (PVN) are the most prominent input areas to LepRb-expressing LH neurons.

Effects of N-acetyl-l-cysteine on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens.

The purpose of this study was to discuss the effects of N-acetyl-l-cysteine (NAC) on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens in different periods. A total of 120 Hy-Line variety brown laying hens (12 weeks old) were randomly assigned to 4 groups with 6 replicates. The control group (C group) (22 ± 1 °C) received a basal diet, the NAC-treated group (N group) (22 ± 1 °C) received a basal diet with 1000 mg/kg NAC, and 2 heat-stressed groups (36 ± 1 °C for 10 h per day and 22 ± 1 °C for the remaining time) were fed a basal diet (HS group) or a basal diet with 1000 mg/kg NAC (HS + N group) for 21 consecutive days. The influence of NAC on histologic changes, oxidative stress and proinflammatory cytokine production was measured and analysed in hens with heat stress-induced hypothalamic changes. NAC effectively alleviated the hypothalamic morphological changes induced by heat stress. In addition, NAC attenuated the activity of the Nf-κB pathway activated by heat stress and decreased the expression of the proinflammatory cytokines IL-6, IL-18, TNF-α, IKK, and IFN-γ. In addition, NAC treatment regulated the expression of HO-1, GSH, SOD2 and PRDX3 by regulating the activity of Nrf2 at different time points to resist oxidative stress caused by heat exposure. In summary, dietary NAC may be an effective candidate for the treatment and prevention of heat stress-induced hypothalamus injury by preventing Nf-κB activation and controlling the Nrf2 pathway.

Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.

In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.

Central administration of insulin-like growth factor-2 suppresses food intake in chicks.

Insulin-like growth factor (IGF)-2 is a multifunctional hormone with structural and functional similarity to IGF-1 in mammals and chickens. We previously showed that intracerebroventricular administration of IGF-1 suppresses food intake in chicks. Also, central administration of IGF-2 suppresses food intake in rats. In the present study, we evaluated whether IGF-2 is involved in the regulation of food intake in chicks. We also examined the effects of fasting on the mRNA levels of IGF binding proteins (IGFBPs) in the liver and hypothalamus, because IGFBPs bind IGF-1 and -2 in plasma and block their binding to the receptors, and locally expressed IGFBPs also influence IGFs binding to the receptors in mammals. Intracerebroventricular administration of IGF-2 significantly suppressed food intake in chicks. The mRNA levels of IGFBPs in the hypothalamus were not affected by six hours of fasting. On the other hand, six hours of fasting markedly increased the mRNA levels of hepatic IGFBP-1 and -2 (5.47- and 6.95-fold, respectively). The mRNA levels of IGFBP-3 were also significantly increased (1.36-fold) by six hours of fasting, whereas the mRNA levels of IGF-2, IGFBP-4, and -5 were unchanged. These findings suggest that circulating IGF-2 may be involved in satiety signals, but its physiological role may be regulated by IGFBPs production in the liver in chicks.

Genetic regulation and heritability of miRNA and mRNA expression link to phosphorus utilization and gut microbiome.

Improved utilization of phytates and mineral phosphorus (P) in monogastric animals contributes significantly to preserving the finite resource of mineral P and mitigating environmental pollution. In order to identify pathways and to prioritize candidate genes related to P utilization (PU), the genomic heritability of 77 and 80 trait-dependent expressed miRNAs and mRNAs in 482 Japanese quail were estimated and eQTL (expression quantitative trait loci) were detected. In total, 104 miR-eQTL (microRNA expression quantitative traits loci) were associated with SNP markers (false discovery rate less than 10%) including 41 eQTL of eight miRNAs. Similarly, 944 mRNA-eQTL were identified at the 5% False discovery rate threshold, with 573 being cis-eQTL of 36 mRNAs. High heritabilities of miRNA and mRNA expression coincide with highly significant eQTL. Integration of phenotypic data with transcriptome and microbiome data of the same animals revealed genetic regulated mRNA and miRNA transcripts (SMAD3, CAV1, ENNPP6, ATP2B4, miR-148a-3p, miR-146b-5p, miR-16-5p, miR-194, miR-215-5p, miR-199-3p, miR-1388a-3p) and microbes (Candidatus Arthromitus, Enterococcus) that are associated with PU. The results reveal novel insights into the role of mRNAs and miRNAs in host gut tissue functions, which are involved in PU and other related traits, in terms of the genetic regulation and inheritance of their expression and in association with microbiota components.

Understanding Transcriptomic and Serological Differences between Forced Molting and Natural Molting in Laying Hens.

Molting is natural adaptation to climate change in all birds, including chickens. Forced molting (FM) can rejuvenate and reactivate the reproductive potential of aged hens, but the effect of natural molting (NM) on older chickens is not clear. To explore why FM has a dramatically different effect on chickens compared with NM, the transcriptome analyses of the hypothalamus and ovary in forced molted and natural molted hens at two periods with feathers fallen and regrown were performed. Additionally, each experimental chicken was tested for serological indices. The results of serological indices showed that growth hormone, thyroid stimulating hormone, and thyroxine levels were significantly higher (p < 0.05) in forced molted hens than in natural molted hens, and calcitonin concentrations were lower in the forced molted than in the natural molted hens. Furthermore, the transcriptomic analysis revealed a large number of genes related to disease resistance and anti-aging in the two different FM and NM periods. These regulatory genes and serological indices promote reproductive function during FM. This study systematically revealed the transcriptomic and serological differences between FM and NM, which could broaden our understanding of aging, rejuvenation, egg production, and welfare issues related to FM in chickens.

Dietary stevioside supplementation increases feed intake by altering the hypothalamic transcriptome profile and gut microbiota in broiler chickens.

BACKGROUND: Stevioside (STE) is a widely used sweetener. Despite the fact that chickens are insensitive to sweetness, dietary STE supplementation could increase the feed intake of broiler chickens. Stevioside might regulate the feeding behavior through functional mechanisms other than its high-potency sweetness. The present study was aimed to elucidate the potential sweetness-independent mechanism of an STE-induced orexigenic effect using the broiler chicken and considering the hypothalamic transcriptome profile and gut microbiome. RESULTS: The analysis of RNA-Seq identified 398 differently expressed genes (160 up-regulated and 238 down-regulated) in the hypothalamus of the STE-supplemented group compared with the control group. Cluster analysis revealed several appetite-related genes were differentially expressed, including NPY, NPY5R, TSHB, NMU, TPH2, and DDC. The analysis of 16S rRNA sequencing data also indicated that dietary STE supplementation increased the relative abundance of Lactobacillales, Bacilli, Lactobacillus, and Lactobacillaceae. Meanwhile, the proportion of Ruminococcaceae, Lachnospiraceae, Clostridia, and Clostridiales was decreased after dietary supplementation with STE. CONCLUSION: Dietary STE supplementation promoted feed intake through the regulation of the hypothalamic neuroactive ligand-receptor interaction pathway and the alteration of intestinal microbiota composition. This study provides valuable information about the sweetness-independent mechanism of the STE-induced orexigenic effect using the broiler chicken (which is insensitive to sweetness) as the animal model. © 2020 Society of Chemical Industry.

Gene Set Enrichment Analysis of Selenium-Deficient and High-Selenium Rat Liver Transcript Expression and Comparison With Turkey Liver Expression.

BACKGROUND: Better biomarkers of selenium (Se) status and a better understanding of toxic Se biochemistry are needed to set safe dietary upper limits. In previous studies, differential expression (DE) of individual liver transcripts in rats and turkeys failed to identify a single transcript that was consistently and significantly (q < 0.05) altered by high Se. OBJECTIVES: To evaluate the effect of Se status on rat liver transcript expression data at the level of gene sets, and to compare transcript expression in rats with that in turkeys to identify common regulated transcripts. METHODS: Gene set enrichment analysis (GSEA) was conducted on liver from weanling rats fed an Se-deficient basal diet (0.005 µg Se/g) supplemented with 0, 0.24 (Se-adequate), 2, or 5 µg Se/g diet as selenite for 28 d. In addition, transcript expression was compared with liver expression in turkeys fed 0, 0.4, 2, or 5 µg Se/g diet as selenite. RESULTS: Se deficiency significantly downregulated the rat selenoprotein gene set but also upregulated gene sets for a variety of pathways, processes, and disease states. GSEA of 2 compared with 0.24 µg Se/g found no significantly up- or downregulated gene sets, showing that 2 µg Se/g is not particularly toxic to the rat. GSEA analysis of 5 compared with 0.24 µg Se/g transcripts, however, found 27 significantly upregulated gene sets for a wide variety of conditions. Cross-species GSEA comparison of transcript expression, however, identified no common gene sets significantly and consistently regulated by high Se in rats and turkeys. In addition, comparison of individual marginally significant (unadjusted P < 0.05) DE transcripts between rats and turkeys also failed to find common transcripts. CONCLUSIONS: The dramatic increase in significant liver transcript DE and GSEA gene sets in rats fed 5 compared with 2 µg Se/g clearly appears to be a biomarker for Se toxicity, albeit not Se-specific. These analyses, however, failed to identify specific transcripts or pathways, biological states, or processes that were directly linked with high Se status, strongly indicating that adaptation to high Se lies outside transcriptional regulation.

Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production.

BACKGROUND: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. RESULTS: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells. CONCLUSIONS: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone more detrimental in HEPH and estradiol stimulatory effects more prominent in LEPH. Responsiveness to thyroid hormone and estradiol may be due to desensitization to thyroid hormone and estradiol in LEPH and HEPH, respectively, due to up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the regulatory role of the HPT axis and beta-estradiol on ovulation rates in turkey hens.

Effects of taurine supplementation on productive performance, nutrient digestibility and gene expression of nutrient transporters in quails reared under heat stress.

This study was conducted to examine the effects of dietary taurine supplementation on productive performance, nutrient digestibility, antioxidant status, and the gene expression of ileal nutrient transporters in laying quails reared under heat stress (HS). One hundred and eighty laying Japanese quails (Coturnix coturnix japonica) were fed a basal diet or basal diet supplemented with either 2.5 or 5 g of taurine per kg of diet, and reared at either 22 ± 2 °C for 24 h/d (thermoneutral, TN) or 34 ± 2 °C for 8 h/d (HS) for 12 weeks. The quails reared under HS consumed less feed, produced less egg, and had lower dry matter, organic matter and crude protein apparent digestibilities compared with the quails reared under the TN condition (P = 0.001). However, increasing taurine concentrations in the diet improved feed intake and egg production (P = 0.001), but also the apparent digestibilities (P ≤ 0.027) in quails reared under HS. The greater doses (5 g/kg) of taurine resulted in more responses. The quails reared under HS had greater serum and liver MDA concentrations (P = 0.0001) which decreased with dietary taurine supplementations, particularly greater doses. The gene expressions of ileal PEPT1, EAAT3, CAT1, CAT2, SGLT1, SGLT5, GLUT2, and GLUT5 decreased under HS conditions (P = 0.001). However, supplementing taurine, in a dose-dependent fashion, to the diet of quails reared under HS resulted in increases in the gene expressions of the transporters (P < 0.05) except for CAT1. The results of the present work showed that taurine supplementation, particularly with greater doses (5 g/kg), to the diet of laying quails kept under HS acts as alleviating negative effects of HS, resulting in improvements in productive performance and nutrient digestion, and also upregulation of ileal nutrient transporters.

Sanguinarine modulate gut microbiome and intestinal morphology to enhance growth performance in broilers.

Sanguinarine is a bioactive compound as a quaternary benzophenanthridine alkaloid from plant of the Macleaya cordata, Papaveraceae family. The present study was conducted to investigate the effects of dietary sanguinarine supplementation on growth performance, serum biochemistry parameters, intestinal mucosal morphology and gut microbiome in yellow feathered broilers. Two hundred and seventy 1-d-old female broilers were randomly assigned to 3 treatments ① Basal diet (NG); ② Basal diet containing bacitracin methylene disalicylate (50mg/Kg diet) (ANT); ③ Basal diet containing sanguinarine (0.7 mg/ kg of feed) (SAG). The statistical results showed that dietary sanguinarine supplementation enhanced growth performance and decreased glucose, uric acid as well as urea nitrogen levels of broilers at 28d of age (P<0.05). The 16S rRNA gene sequence analysis revealed that sanguinarine significantly decreased the species from the phyla Bacteroidetes, and increased the species from phyla Firmicutes. Moreover, dietary sanguinarine supplementation improved mucosal morphology to achieve higher ratio of intestinal villus height to crypt depth (P < 0.05), and decreased the concentrations of TNF-α and IL-4 in jejunum mucosal. This study demonstrated that sanguinarine supplementation in the diet of yellow feathered broilers improved intestinal morphology and microbiota community structure to promote growth performance on 1-28d.

Transcriptome analysis reveals a molecular understanding of nicotinamide and butyrate sodium on meat quality of broilers under high stocking density.

BACKGROUND: In recent years, increased attention has been focused on breast muscle yield and meat quality in poultry production. Supplementation with nicotinamide and butyrate sodium can improve the meat quality of broilers. However, the potential molecular mechanism is not clear yet. This study was designed to investigate the effects of supplementation with a combination of nicotinamide and butyrate sodium on breast muscle transcriptome of broilers under high stocking density. A total of 300 21-d-old Cobb broilers were randomly allocated into 3 groups based on stocking density: low stocking density control group (L; 14 birds/m2), high stocking density control group (H; 18 birds/m2), and high stocking density group provided with a combination of 50 mg/kg nicotinamide and 500 mg/kg butyrate sodium (COMB; 18 birds/m2), raised to 42 days of age. RESULTS: The H group significantly increased cooking losses, pH decline and activity of lactate dehydrogenase in breast muscle when compared with the L group. COMB showed a significant decrease in these indices by comparison with the H group (P < 0.05). The transcriptome results showed that key genes involved in glycolysis, proteolysis and immune stress were up-regulated whereas those relating to muscle development, cell adhesion, cell matrix and collagen were down-regulated in the H group as compared to the L group. In contrast, genes related to muscle development, hyaluronic acid, mitochondrial function, and redox pathways were up-regulated while those associated with inflammatory response, acid metabolism, lipid metabolism, and glycolysis pathway were down-regulated in the COMB group when compared with the H group. CONCLUSIONS: The combination of nicotinamide and butyrate sodium may improve muscle quality by enhancing mitochondrial function and antioxidant capacity, inhibiting inflammatory response and glycolysis, and promoting muscle development and hyaluronic acid synthesis.

Effects of lysine and methionine in a low crude protein diet on the growth performance and gene expression of immunity genes in broilers.

Globally, the poultry industry is 1 of the most advanced livestock industries. Feed contributes to the biggest proportion (65-70%) of the production cost. Most feed ingredients in Malaysia are imported, which contributes to the high food bill annually, and alternative feed formulation may help decrease the cost of poultry feed. Feed formulation are improved to efficiently meet the dietary requirements of the broilers and 1 of the ways is by reducing the level of crude protein in the diet while supplementing essential amino acids. In this study, the effects of methionine and lysine, which are the 2 most limiting amino acids in the chicken diet, were supplemented in a low crude protein diet, and its effects on the growth and expression of immunity genes such as MUC2, SLC, GAL6, and LEAP-2 were studied. A total of 300 Cobb500 broilers were tested with 10 different dietary treatments. Experimental treatment diets consist of high, standard, and low levels of methionine and lysine in the diet with reduced crude protein. The control group consists of diet with standard levels of lysine, methionine, and crude protein as recommended for Cobb500 broilers. Ribonucleic acid was extracted from the jejunum, spleen, and liver for gene expression analysis which was performed with real-time polymerase chain reaction using SYBR Green chemistry. Results of the growth performance at 6 wk showed improved feed conversion ratio when lysine was increased by 0.2% in a low crude protein diet at 1.96 ± 0.11. Gene expression of MUC2 gene in the jejunum showed a significant increase across all experimental diets with the treatment with higher lysine in low crude protein diet with the highest increase of 3.8 times as compared with the control diet. The other genes expressed in the spleen and liver were mostly downregulated. It was concluded that supplementation of high lysine with standard methionine in a low crude protein diet performed better in terms of lowest feed conversion ratio and high upregulation of MUC2 gene.

Maternal betaine supplementation decreases hepatic cholesterol deposition in chicken offspring with epigenetic modulation of SREBP2 and CYP7A1 genes.

Maternal betaine was reported to regulate offspring hepatic cholesterol metabolism in mammals. However, it is unclear whether and how feeding betaine to laying hens affects hepatic cholesterol metabolism in offspring chickens. Rugao yellow-feathered laying hens (n = 120) were fed basal or 0.5% betaine-supplemented diet for 28 D before the eggs were collected for incubation. Maternal betaine significantly decreased the hepatic cholesterol content (P < 0.05) in offspring chickens. Accordingly, the cholesterol biosynthetic enzymes, sterol regulator element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase, were decreased, while cholesterol-7alpha-hydroxylase (CYP7A1), which converts cholesterol to bile acids, was increased at both mRNA and protein levels in betaine-treated offspring chickens. Hepatic mRNA and protein expression of low-density lipoprotein receptor was significantly (P < 0.05) increased, while the mRNA abundance of cholesterol acyltransferase 1 (ACAT1) that mediates cholesterol esterification was significantly (P < 0.05) decreased in the betaine group. Meanwhile, hepatic protein contents of DNA methyltransferases 1 and betaine homocysteine methyltransferase were increased (P < 0.05), which was associated with modifications of CpG methylation on affected cholesterol metabolic genes. Furthermore, the level of CpG methylation on gene promoters was increased (P < 0.05) for sterol regulator element-binding protein 2 and abundance of cholesterol acyltransferase 1 yet decreased (P < 0.05) for cholesterol-7alpha-hydroxylase. These results indicate that maternal betaine supplementation significantly decreases hepatic cholesterol deposition through epigenetic regulation of cholesterol metabolic genes in offspring juvenile chickens.

Dietary betaine improves egg-laying rate in hens through hypomethylation and glucocorticoid receptor-mediated activation of hepatic lipogenesis-related genes.

In avian species, liver lipid metabolism plays an important role in egg laying performance. Previous studies indicate that betaine supplementation in laying hens improves egg production. However, it remains unclear if betaine improves laying performance by affecting hepatic lipid metabolism and what mechanisms are involved. We fed laying hens a 0.5% betaine-supplemented diet for 4 wks to investigate its effect on hepatic lipids metabolism in vivo and confirmed its mechanism via in vitro experiments using embryonic chicken hepatocytes. Results showed that betaine supplemented diet enhanced laying production by 4.3% compared with normal diet, accompanied with increased liver and plasma triacylglycerol concentrations (P < 0.05) in hens. Simultaneously, key genes involved in hepatic lipid synthesis, such as sterol regulatory element binding protein 1 (SREBP-1), fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase 1 (SCD1) were markedly upregulated at the mRNA level (P < 0.05). Western blot results showed that SREBP-1 and SCD1 protein levels were also increased (P < 0.05). Moreover, mRNA expression of main apolipoprotein components of yolk-targeted lipoproteins, apolipoprotein B (ApoB) and apolipoprotein-V1 (ApoV1), in addition to microsomal triglyceride transfer proteins, which is closely related to the synthesis and release of very-low density lipoprotein, were also markedly elevated (P < 0.05). Methylated DNA immunoprecipitation combined with PCR detects reduction of methylation levels in certain regions of the above gene promoters. Chromatin immunoprecipitation PCR assays showed increased binding of glucocorticoid receptor (GR) to SREBP1 and ApoB gene promoters. Similar results of ApoV1 gene expression were obtained from cultured hepatocytes treated with betaine. Additionally, betaine increased the expression of GR and some genes involved in methionine cycle in vitro. These results suggest that betaine supplementation could alter the expression of liver lipid synthesis and transport-related genes by modifying the methylation status and GR binding on their promoter and hence promote the synthesis and release of yolk precursor substances in the liver.

Fetal betaine exposure modulates hypothalamic expression of cholesterol metabolic genes in offspring cockerels with modification of promoter DNA methylation.

In documents, maternal betaine modulates hypothalamic cholesterol metabolism in chicken posthatchings, but it remains unclear whether this effect can be passed on by generations. In present study, eggs were injected with saline or betaine at 2.5 mg/egg, and the hatchlings (F1) were raised under the same condition until sexual maturation. Both the control group and the betaine group used artificial insemination to collect sperm from their cockerels. Fertilized eggs were incubated, and the hatchlings of the following generation (F2) were raised up to 64 D of age. F2 cockerels in betaine group showed significantly (P < 0.05) lower body weight, which was associated with significantly decreased (P < 0.05) hypothalamic content of total cholesterol and cholesterol ester. Concordantly, hypothalamic expression of cholesterol biosynthetic genes, SREBP2 and HMGCR, were significantly downregulated (P < 0.05), together with cholesterol conversion-related and excretion-related genes, CYP46A1 and ABCA1. These changes coincided with a significant downregulation in mRNA expression of regulatory neuropeptides including brain-derived neurotrophic factor, neuropeptide Y, and corticotropin-releasing hormone. Moreover, genes involved in methyl transfer cycle were also modified. Betaine homocysteine methyltransferase (P < 0.05) was downregulated, yet DNA methyltransferase1 tended to be upregulated (P = 0.06). S-adenosyl methionine/S-adenosylhomocysteine ratio was higher in the hypothalamus of betaine-treated F2 cockerels, which was associated with significantly modified CpG methylation on the promoter of those affected genes. These results suggested that betaine might regulate central cholesterol metabolism and hypothalamic expression of genes related to brain function by altering promoter DNA methylation in F2 cockerels.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

Effects of available phosphorus source and concentration on performance and expression of sodium phosphate type IIb cotransporter, vitamin D-1α-hydroxylase, and vitamin D-24-hydroxylase mRNA in broiler chicks.

This experiment was conducted to examine the effect of 2 phosphorus (P) sources on broiler performance to day 14. The P bioavailability was estimated using bird performance and tibia ash measurements, whereas P digestibility, intestinal P transporter, kidney vitamin D-1α-hydroxylase, and vitamin D-24-hydroxylase mRNA abundances were also determined. Slope regression analysis was used to determine the bioavailability of dicalcium phosphate (Dical P) and nanocalcium phosphate (Nano P) with dietary available P (AvP) set to 0.20% P (control) using AvP from the major ingredients and Dical P. The experimental treatments were achieved by supplementation with either Dical P or Nano P to generate 0.24, 0.28, 0.32, and 0.36% AvP. A total of 648-day-old unsexed broiler chicks were divided into 72 birds per treatment (8 replicate cages of 9 birds). Slope regression analysis showed positive linear relationships between BW, feed intake (FI), tibia ash weight (TAW), and tibia ash percentage (TAP) with dietary Dical P and Nano P levels. Comparisons between regression slopes for Dical P and Nano P fed birds were not significantly different for BW, feed intake, tibia ash weight, and tibia ash percentage, indicating similar P bioavailability from Dical P and Nano P. There were interactions between P source and AvP for feed efficiency (FE) and apparent ileal P digestibility (AIPD). Dicalcium phosphate had greater FE than Nano P at 0.28% AvP and greater AIPD than Nano P at 0.24% AvP. The addition of AvP from Dical P and Nano P resulted in reduced sodium phosphate cotransporter mRNA abundance in the duodenum in a dose-dependent response. In the kidney, vitamin D-1α-hydroxylase mRNA abundance was greater at 0.36% Nano P compared with control, but there was no difference with Dical P. There was no difference in vitamin D-24-hydroxylase mRNA abundance between control and supplementation with Nano P or Dical P. In conclusion, Nano P and Dical P had the same bioavailability but had different effects on gene expression.