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.

MiR-144-3p targets STC1 to activate PI3K/AKT pathway to induce cell apoptosis and cell cycle arrest in selenium deficiency broilers.

Selenium (Se) is an indispensable trace element in vertebrate. Se deficiency can damage the immune system. Studies have shown that Se deficiency can cause immune organ damage by regulating the expression of microRNA. Bursa of Fabricius is a special immune organ in poultry. In order to explore the mechanism of bursa of Fabricius injury caused by Se deficiency and the role of miRNA in this process. Firstly, we established the Se deficient model of broilers in vivo and found that Se deficiency could induce apoptosis and cell cycle arrest of bursa of Fabricius cells through Phosphoinositide 3-kinase (PI3K)/Protein Kinase B (AKT) pathway. Secondly, we inferred miRNA (miR-144-3p) and target gene Stanniocalcin 1 (STC1) that may regulate PI3K/AKT pathway through biological analysis system, and further predicted and determined the targeting relationship between them through dual luciferase, it was found that miR-144-3p was highly expressed in the process of cell apoptosis and cell cycle arrest induced by Se deficiency. Finally, in order to further understand whether miR-144-3p/STC1 axis is involved in the process, miR-144-3p knockdown and overexpression experiments were carried out, it was found that miR-144-3p inhibitor can reduce the occurrence of cell apoptosis and cell cycle arrest. In conclusion, Se deficiency can induce apoptosis and cell cycle arrest of bursa of Fabricius in Broilers by up regulating miR-144-3p targeting STC1 and activating PI3K/AKT pathway, leading to injury of bursa of Fabricius in broilers.

Cadmium induced Fak -mediated anoikis activation in kidney via nuclear receptors (AHR/CAR/PXR)-mediated xenobiotic detoxification pathway.

Cadmium (Cd) is a toxic heavy metal of considerable toxicity, possessing a serious environmental problem that threatening food safety and human health. However, the underlying mechanisms of Cd-induced nephrotoxicity and detoxification response remain largely unclear. Cd was administered at doses of 35, 70, and 140 mg/kg diet with feed for 90 days and produced potential damage to chickens' kidneys. The results showed that Cd exposure induced renal anatomical and histopathological injuries. Cd exposure up-regulated cytochrome P450 enzymes (CYP450s), activated nuclear xenobiotic receptors (NXRs) response, including aryl hydro-carbon receptor (AHR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) by low and moderate doses of Cd, and induced an increase in CYP isoforms expression. Cd exposure down-regulated phase II detoxification enzymes (glutathione-S-transferase (GST), glutathione peroxidase (GSH-PX) activities, and glutathione (GSH) content), and GST isoforms transcription . Furthermore, ATP-binding cassette (ABC) transporters, multidrug resistance protein (MRP1), and P-glycoprotein (P-GP) levels were elevated by low dose, but high dose inhibited the P-GP expression. Activation of detoxification enzymes lost their ability of resistance as increasing dose of Cd, afterwards brought into severe renal injury. Additionally, Cd suppressed focal adhesion kinase (Fak) and integrins protein expression as well as activated extrinsic pathway and intrinsic pathways, thereby producing anoikis. In conclusion, these results indicated that Cd induced Fak-mediated anoikis activation in the kidney via nuclear receptors (AHR/CAR/PXR)-mediated xenobiotic detoxification pathway.

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.

A Green and Blue Monochromatic Light Combination Therapy Reduces Oxidative Stress and Enhances B-Lymphocyte Proliferation through Promoting Melatonin Secretion.

Artificial illumination may interfere with biological rhythms and distort physiological homeostasis in avian. Our previous study demonstrated that 660 nm red light exacerbates oxidative stress, but a combination of green and blue lights (G→B) can improve the antibody titer in chickens compared with single monochromatic light. Melatonin acts as an antioxidant which is a critical signaling to the coordination between external light stimulation and the cellular response from the body. This study further clarifies the potential role of melatonin in monochromatic light combination-induced bursa B-lymphocyte proliferation in chickens. A total of 192 chicks were exposed to a single monochromatic light (red (R), green (G), blue (B), or white (W) lights) or various monochromatic light combinations (B→G, G→B, and R→B) from P0 to P42. We used qRT-PCR, MTT, western blotting, immunohistochemistry, and Elisa to explore the effect of a combination of monochromatic light on bursa B-lymphocytes and its intracellular signal pathways. With consistency in the upregulation in melatonin level of plasma and antioxidant enzyme ability, we observed increases in organ index, follicle area, lymphocyte density, B-lymphocyte proliferation, PCNA-positive cells, and cyclin D1 expression in bursa of the G→B group compared with other light-treated groups. Melatonin bound to Mel1a and Mel1c and upregulated p-AKT, p-PKC, and p-ERK expression, thereby activating PI3K/AKT and PKC/ERK signaling and inducing B-lymphocyte proliferation. Overall, these findings suggested that melatonin modulates a combination of green and blue light-induced B-lymphocyte proliferation in chickens by reducing oxidative stress and activating the Mel1a/PI3K/AKT and Mel1c/PKC/ERK pathways.

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.

Supplemental 25-hydroxycholecalciferol Alleviates Inflammation and Cardiac Fibrosis in Hens.

Broiler breeder hens with efficient feed conversion rate under restricted feed intake (R-hens) or allowed unlimited access to feed (Ad-hens) progressed with cardiac functional failure and suffered early sudden death. A supplement of 69 µg 25-hydroxycholecalciferol (25-OH-D3)/kg feed improved heart health and rescued livability in both R- and Ad-hens throughout laying stage (26-60 wks). Improvements occurred through cardiac hypertrophic remodeling, reduced arrhythmias, and pathological cues. Here, we further demonstrated consistently decreased circulating and cardiac IL-6 and IL-1ß levels in conjunction with reduced cardiac chemoattraction and leukocyte infiltration by 25-OH-D3 in Ad-hens and in R-hens at later time points (35 and 47 wks) (p < 0.05). Supplemental 25-OH-D3 also ameliorated cardiac fibrosis, endoplasmic reticulum (ER) stress, and autophagy, mostly in Ad-hens, as both collagen content and expression of COL3A1, as well as CCAAT box binding enhancer homologous protein (CHOP) and activating transcription factor 6 (ATF6), were consistently decreased, and suppression of microtubule-associated protein 1 light Chain 3 beta (LC3B) and Sequestosome 1 (SQSTM1) was rescued at 35 and 47 wks (p < 0.05). Vitamin D receptor-NF-κB signaling was shown to mediate these beneficial effects. The present results demonstrate that ER stress and autophagic processes along the sequence from inflammation to fibrotic changes contribute to pathological cardiac remodeling and functional compromise by Ad-feed intake. 25-OH-D3 is an effective anti-inflammatory and anti-fibrotic supplement to ameliorate cardiac pathogenesis in broiler breeder hens.

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.

The antagonistic effect of selenium on lead-induced necroptosis via MAPK/NF-κB pathway and HSPs activation in the chicken spleen.

Recent studies identified a novel programmed and regulated cell death that was characterized by a necrotic cell death morphology, termed necroptosis. Lead (Pb) is known as a persistent inorganic environmental pollutant that affects the health of humans and animals worldwide. However, there are no detailed reports of Pb-induced necroptosis of immune tissue. Selenium (Se) is a trace element that antagonizes the toxicity of heavy metals. Here, chickens were randomly divided into four groups, treated with Pb ((CH3OO)2Pb, 150 mg/kg) and/or Se (Na2SeO3, 2 mg/kg), aim to study the effect and mechanism of necroptosis in Pb-induced spleen injury and the antagonistic effects of Se on Pb toxicity. Our results showed that Pb exposure evidently increased the accumulation of Pb in spleen and caused necroptosis by upregulating the expression of RIP1, RIP3 and MLKL, and decreasing Caspase8 expression. Meanwhile, Pb treatment inhibited the activities of SOD, GPX, and CAT, caused the accumulation of NO and MDA, and induced oxidative stress, which promoted the expression of MAPK/NF-κB pathway genes (ERK, JNK, P38, NF-κB and TNF-α) and activated HSPs (HSP27, HSP40, HSP60, HSP70 and HSP90). However, the increased content of Pb in spleen and Pb-caused necroptosis were inhibited by Se cotreatment. Overall, we conclude that Se can prevent Pb-induced necroptosis by restoring antioxidant functions and blocking the MAPK/NF-κB pathway and HSPs activation in chicken spleen.

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.

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.

Effects of fermented feed on growth performance, immune response, and antioxidant capacity in laying hen chicks and the underlying molecular mechanism involving nuclear factor-κB.

This study investigated the effects of fermented-feed diets on growth performance, immune status, and antioxidant responses in laying hen chicks and the underlying molecular mechanism, specifically, the role of the nuclear factor-κB (NF-κB) signaling pathway. A total of 80 healthy 14-day-old laying hen chicks were randomly divided into 4 treatments: basal diet (CON); basal diet supplemented with 7.5% fermented feed (FD); FD diet plus the NF-κB inhibitor BAY 11-7082 (FD + BAY); and FD diet plus the NF-κB inhibitor JSH-23 (FD + JSH). The NF-κB inhibitors were administered by intraperitoneal injection. The experiment lasted 21 D. Fermented feed supplementation significantly increased the body weight and average body weight gain of laying hen chicks but significantly decreased the feed conversion ratio. Additionally, fermented feed supplementation significantly increased mitogen-activated T-cell and B-cell proliferation in the peripheral blood, as well as elevated the serum concentrations of interleukin (IL)-1, IL-2, IL-4, IL-6, and tumor necrosis factor (TNF-α); however, NF-κB inhibition significantly reduced T-cell proliferation and serum IL-1, IL-6, and TNF-α levels. The levels of IgA, IgG, IgM, and Newcastle disease virus antibody in the serum were significantly increased by the addition of fermented feed. Furthermore, fermented feed supplementation significantly improved antioxidant function, as indicated by the increases of total antioxidant capacity, total superoxide dismutase activity, and glutathione peroxidase activity and the decrease of malonaldehyde level. However, NF-κB inhibition reversed these changes. Western blot analysis showed that fermented feed treatment increased splenic IκB kinase ß and NF-κB protein levels, whereas these increases were prevented by NF-κB inhibition. In conclusion, fermented feed improves the growth performance, immune function, and antioxidant capacity of laying hen chicks. Fermented feed-induced modulation of T-cell proliferation, T helper type 1 and T helper type 2 cytokine production, and antioxidation is associated with NF-κB activation.

Short chain fatty acids may improve hepatic mitochondrial energy efficiency in heat stressed-broilers.

The present study was conducted to investigate the effects of four dietary fat types and two environmental temperatures on the hepatic mitochondrial energetic in male broilers exposed to heat stress. The birds were kept in two separate rooms at 24 °C or 36 °C from 32 to 42 d of age with four experimental groups in each room. The birds fed on the diets supplemented containing rich sources of long-chain saturated fatty acids (beef tallow), middle-length-chain saturated FA (coconut oil), monounsaturated FA (olive oil), or polyunsaturated FA (soybean oil) for ten days. At 36 °C, the highest body weight and lowest feed conversion ratio were recorded in the birds fed on the diets supplemented with coconut oil or beef tallow. Temperature and fat type significantly affected the activities of the mitochondrial electron transport chain complexes (P < 0.01). There was a significant interaction between the temperature and fat type (P < 0.01). Generally, electron transport chain complexes I-V enzymatic activities were decreased at 36 °C. The coconut oil-fed birds showed the highest complex I activity at both temperatures. The beef tallow-fed broilers showed the lowest complex II activity at 24 °C. In birds exposed to 36 °C, complex II activity was higher for birds fed saturated coconut oil or beef tallow than those feeding the unsaturated olive oil or soybean oil-supplemented diets. At 24 °C, the highest and lowest complex III activities were recorded for the coconut oil- and beef tallow-supplemented diets, respectively. At 36 °C, the activity of complex III was coconut oil > beef tallow > olive oil > soybean oil. At 24 °C, complex IV activity was highest in coconut oil- or soybean oil-fed broilers; and at 36 °C, complex IV showed the lowest activity in soybean oil-fed birds. The highest complex IV activity was observed in coconut oil-fed chickens followed by olive oil-fed and beef tallow-fed birds, respectively. At 24 or 36 °C, the highest and lowest complex V activity was observed in coconut oil-fed and soybean oil-fed chickens, respectively. ATP concentration and mitochondrial membrane potential were in the order of coconut oil > beef tallow > olive oil > soybean oil at both temperatures. Temperature and fat type significantly affected the avANT mRNA concentration. Exposure of broilers to 36 °C generally decreased the mRNA expression of avANT, with beef tallow- or coconut oil-supplemented birds showing a lower avANT mRNA expression than those receiving olive oil- or soybean oil-supplemented diets. These findings provide further information on the use of fat sources in the diet of heat stressed-broilers.