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ABSTRACT: Oxidative stress is a frequent concern in the breeding of laying hens, and limit the healthy development of poultry. Dexamethasone (DXM) has been demonstrated to induce oxidative stress. Conversely, betaine is an alkaloid with a potent antioxidant activity. The study was designed to investigate the ameliorative effect of betaine on DXM-induced oxidative stress in laying hens. The results revealed that DXM treatment significantly decreased laying rate, shell strength, albumen height, Haugh unit, egg weight, folk weight and albumen weight, alongside increased malondialdehyde (MDA) and decreased total antioxidant capacity (T-AOC) in serum and liver (P < 0.05). In contrast, dietary betaine addition reversed those parameters mentioned above (P < 0.05). Hepatic RNA-seq analysis showed that there existed 110 up- and 88 down-regulated genes in DXM group when compared with the control. Meanwhile there were 117 upregulation and 169 downregulation genes in BT group when compared with DXM group. Besides, we found that dietary betaine addition significantly down-regulated cell adhesion molecules, glycerolipid metabolism and glycolysis gluconeogenesis pathways. In addition, a total of 44 and 94 differential metabolites were identified respectively from Con vs. DXM and DXM vs BT. More importantly, dietary betaine addition significantly increased the levels of pantothenic acid, gamma-Aminobutyric acid, equol and choline, all of which were related to antioxidant and anti-inflammatory properties. Furthermore, gut microbiota analysis indicated that the Chao and Observed_species indexes were remarkably higher in BT group (P

ABSTRACT: The hyperplasia and hypertrophy of preadipocytes were closely related to lipid deposition in animals. Butyric acid was reported to be involved in lipid metabolism. The aim of the current study was to investigate the effect of butyric acid on the proliferation and differentiation of the immortalized chicken preadipocyte 2 (ICP2). ICP2 were treated respectively with 12mM butyric acid for 48h in proliferation trial and 4mM butyric acid plus 200 μM oleic acid for 3 d in differentiation trial. For the proliferation trial, RNA-seq analysis revealed that 2039 genes were significantly up-regulated and 780 genes were significantly down-regulated with 12 mM butyric acid after 48 h treatment. Concurrently, Cell cycle, DNA replication and p53 signaling pathways were down-regulated in Butyric acid group. More importantly, 12 mM butyric acid restrained the expression of cell proliferation genes such as PCNA, CDK1 and CDK2 in Butyric acid group (P < 0.05), and the protein expression levels of PCNA and CDK1 were also significantly decreased (P < 0.05). The Oil red staining revealed a fewer presence of red fat droplets in ICP2 following treatment with 4 mM butyric acid, accompanied by decreased levels of total cholesterol (TC) and triglycerides (TG). RNA-seq analysis shown that the number of up and down-regulated genes were 2095 and 1042 respectively in OAB group (oleic acid+butyric acid) when compared with OA group (oleic acid). Meanwhile the AMPK signaling pathway, FOXO signaling pathway and focal adhesion were significantly enriched in OAB group. Additionally, 4 mM butyric acid inhibited the expression of lipid differentiation genes including FABP4, C/EBPα, PPARγ and LPL in OAB group (P < 0.05), as well as lipogenesis proteins such as FABP4, C/EBP-α and PPARγ (P < 0.05). In conclusion, 12 mM butyric acid effectively inhibited the proliferation of ICP2 by slowing down cell cycle progression, while 4 mM butyric acid alleviated lipid deposition by reducing the production of lipid droplets through inhibiting the expression of lipid differentiation marker genes and proteins.

ABSTRACT: The late embryonic development of the liver, a major metabolic organ, remains poorly characterized at single cell resolution. Here, we used single-nucleus RNA-sequencing (snRNA-seq) to characterize the chicken liver cells at 2 embryonic development time points (E14 and D1). We uncovered 8 cell types including hepatocytes, endothelial cells, hepatic stellate cells, erythrocytes, cholangiocytes, kupffer cells, mesothelial cells, and lymphocytes. And we discovered significant differences in the abundance of different cell types between E14 and D1. Moreover, we characterized the heterogeneity of hepatocytes, endothelial cells, and mesenchymal cells based on the gene regulatory networks of each clusters. Trajectory analyses revealed 128 genes associated with hepatocyte development and function, including apolipoprotein genes involved hepatic lipid metabolism and NADH dehydrogenase subunits involved hepatic oxidative phosphorylation. Furthermore, we identified the differentially expressed genes (DEGs) between E14 and D1 at the cellular levels, which contribute to changes in liver development and function. These DEGs were significantly enriched in PPAR signaling pathways and lipid metabolism related pathways. Our results presented the single-cell mapping of chick embryonic liver at late stages of development and demonstrated the metabolic changes across the 2 age stages at the cellular level, which can help to further study the molecular development mechanism of embryonic liver.

ABSTRACT: Increased consumption of folic acid is prevalent due to its beneficial effects, but growing evidence emphasizes the side effects pointing to excessive dietary folate intake. The effects of excessive paternal folic acid consumption on offspring and its transgenerational inheritance mechanism have not been elucidated. We hypothesize that excessive folic acid consumption will alter sperm DNA N6-methyladenine (6mA) and 5-methylcytosine (5mC) methylation and heritably influence offspring metabolic homeostasis. Here, we fed roosters either folic acid-control or folic acid-excess diet throughout life. Paternal chronic folic acid excessive supplementation increased hepatic lipogenesis and lipid accumulation but reduced lipolysis both in the roosters and their offspring, which was further confirmed to be induced by one-carbon metabolism inhibition and gene expression alteration associated with the Peroxisome proliferator-activated receptor pathway. Based on the spermatozoal genome-wide DNA methylome identified by Nanopore sequencing, multi-omics association analysis of spermatozoal and hepatic DNA methylome, transcriptome, and metabolome suggested that differential spermatozoal DNA 6mA and 5mC methylation could be involved in regulating lipid metabolism-related gene expression in offspring chickens. This model suggests that sperm DNA N6-methyladenine and 5-methylcytosine methylation were involved in epigenetic transmission and that paternal dietary excess folic acid leads to hepatic lipid accumulation in offspring.

ABSTRACT: Excess abdominal fat reduces carcass yield and feed conversion ratio, thereby resulting in significant economic losses in the poultry industry. Our previous study demonstrated that dietary addition of folic acid reduced fat deposition and changed gut microbiota and short-chain fatty acid. However, whether folic acid regulating abdominal fat deposition was mediated by gut microbiota was unclear. A total of 210 one-day-old broiler chickens were divided into 3 groups including the control (CON), folic acid (FA), and fecal microbiota transplantation (FMT) groups. From 14th day, broiler chickens in CON and FA groups were given perfusion administration with 1 mL diluent daily, while 1 mL fecal microbiota transplantation suspension from FA group prepared before was perfusion in FMT group receiving control diets. The result showed that abdominal fat percentage was significantly lower in FA and FMT groups when compared with CON group (P < 0.05). Morphology analysis revealed that the villus height of jejunum and ileum were significantly higher in FMT group (P < 0.05), and the villus height of jejunum was also significantly higher in FA group (P < 0.05), while the diameter and cross-sectional area (CSA) of adipocytes were significantly decreased in FA and FMT groups when compared with CON group (P < 0.05). Western blot results indicated that the expression levels of FOXO1 and PLIN1 in FMT group were significantly increased (P < 0.05), whereas the expression levels of PPARγ, C/EBPα, and FABP4 were significantly decreased (P < 0.05). Additionally, the Chao1, Observed-species, Shannon and Simpson indexes in FA and FMT groups were significantly higher (P < 0.05), but the microbiota were similar between FMT and FA groups (P < 0.05). LEfSe analysis determined that Lactobacillus, Clostridium and Dehalobacterium were found to be predominant in FA group, while Oscillospira, Shigella, and Streptococcus were the dominant microflora in FMT group. Furthermore, these cecal microbiota were mostly involved in infectious disease, cellular community prokaryotes, cell motility and signal transduction in FA group (P < 0.05), whereas functional capacities involved in signal transduction, cell motility, infectious disease and environment adaptation were enriched significantly of cecal microbiota in FMT group (P < 0.05). In summary, both fecal microbiota transplantation from the broiler chickens of dietary added folic acid and dietary folic acid addition effectively reduced abdominal fat deposition, indicating that the regulatory effect of folic acid on abdominal fat deposition was mediated partly by gut microbiota in broiler chickens.

ABSTRACT: Excess abdominal fat is a common phenomenon in broiler chickens. Gut microbiota could regulate lipid metabolism through their effects on short-chain fatty acids (SCFAs) production. This study was conducted to investigate the potential relationship between abdominal fat development and cecal microorganism populations. Abdominal fat and cecum contents were collected at 3, 7, 14, 21, 28, 35, and 42 d of age. The results showed that abdominal fat weight increased with age. The abdominal fat percentage was higher between 7 and 21 d of age than at 3 d (P < 0.05), and it increased again at 28 to 42 d (P < 0.05). Morphological analysis showed that adipocyte diameter and cross-sectional area (CSA) increased significantly after 14 d of age (P < 0.05). Moreover, gut microbiota analysis indicated that the Chao1 and Shannon indices were higher between 14 and 28 d than at 3 d of age (P < 0.05). Furthermore, LEfse analysis revealed that Faecalibacterium, Anaerotruncus, Anaeroplasma, Subdoligranulum, and Clostridium emerged to become dominant at 14 d. A greater abundance of Bacteroides, Ruminococcus, Dehalobacterium, and Lactobacillus were determined at 28 d when compared with 14 d of age. Parabacteroides, Ochrobactrum, Lactobacillus, Blautia, Alistipes, Dehalobacterium, Odoribacter, and Suuterella were found to be predominant at 42 d. PICRUSt analysis revealed that amino acid metabolism, lipid metabolism, and terpenoids and polyketides metabolism were elevated at 14 d; the immune and digestive systems were significantly developed at 28 d. In addition, cecum propionic acid and butyric acid contents gradually increased (P < 0.05), while the isobutyric acid contents gradually decreased with advancing age (P < 0.05). Correlation analysis among SCFAs, differential genera and abdominal fat suggested that Coprobacillus, Shigella, and Butyricicoccus had negative correlations with propionic acid, butyric acid, and abdominal fat weight, but positive correlations with isobutyric acid. Isobutyric acid was identified as being negatively associated with abdominal fat weight, while the reverse was found for propionic acid and butyric acid. In conclusion, abdominal fat development is correlated with the emergence of specific microbes and d 14 may be a pivotal age for establishing this relationship.

ABSTRACT: Fatty liver syndrome (FLS) is a kind of nutritional metabolic disease in laying hens. Revealing FLS pathogenesis during the early period is what really makes sense for the prevention or nutritional regulation strategies. In the study, 9 healthy or naturally occurring early FLS birds were screened based on visual inspection, liver index and morphologic analysis. Liver and fresh cecal content samples were collected. Then transcriptomic and 16S rRNA technologies are applied to investigate hepatic transcriptome and cecum microbiota composition. Unpaired Student t test and some omics methods were used for statistical analysis. Results showed higher liver weight and index were found in FLS group; morphologic analysis indicated that there existed more lipid droplets in the liver of birds with FLS. Based on DESeq2 analysis, there were 229 up- and 487 down-regulated genes in the FLS group, among which most genes related to de novo fatty acid synthesis were up-regulated such as acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase, and ELOVL fatty acid elongase 6 (ELOVL6). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that pathways associated with lipid metabolism and liver damage were affected. 16S rRNA sequencing analysis of cecum microbiota showed that there was a significant difference between the Con and FLS groups. LEfSe analysis revealed that the relative abundance of Coprococcus, Odoribacter, Collinsella, Turicibacter, YRC22, Enterococcus, Shigella, and Bifidobacterium were down-regulated in the FLS group, whereas the abundance of Bacteroides, Mucispirillum, Butyricicoccus, Campylobacter, Akkermansia, and Clostridium were up-regulated. The KEGG enrichment from differential microbiota suggested that some metabolism-related functions were altered to some extent. Taken together, during the developmental of early fatty liver of laying hens, lipogenesis was enhanced, whereas abnormal metabolism occurs not only in lipid transportation but also in hydrolysis, which caused structural damage to the liver organ. Moreover, the dysbiosis of the cecum microbiota occurred. All of these serve as targets or provide theoretical references for the development of probiotics for fatty liver prevention in laying hens.

ABSTRACT: Coccidia vaccination is a common practice in the poultry industry. However, research is lacking regarding the optimal nutritional support for coccidia vaccinated broilers. In this study, broilers were vaccinated with coccidia oocyst at hatch and were fed with a common starter diet from 1 to 10 d. On d 11, the broilers were randomly assigned to groups in a 4 × 2 factorial arrangement. Briefly, the broilers were fed one of four diets containing 0.6, 0.8, 0.9, and 1.0% of standardized ileal digestible methionine plus cysteine (SID M+C), respectively, from 11 to 21 d. On d 14, the broilers from each diet group were orally gavaged with either PBS (Mock challenge) or Eimeria oocysts. Compared to PBS-gavaged broilers and regardless of dietary SID M+C levels, the Eimeria-gavaged broilers had 1) decreased gain-to-feed ratio (15–21 d, P = 0.002; 11–21 d, P = 0.011); 2) increased fecal oocysts (P < 0.001); 3) increased plasma anti-Eimeria IgY (P = 0.033); and 4) increased intestinal luminal interleukin-10 (IL-10; duodenum, P = 0.039; jejunum, P = 0.018) and gamma interferon (IFN-γ; duodenum, P < 0.001; jejunum, P = 0.017). Regardless of Eimeria gavage, broilers fed 0.6% SID M+C had decreased (P

ABSTRACT: The liver undergoes a slow process for lipid deposition during chick embryonic period. However, the underlying physiological and molecular mechanisms are still unclear. Therefore, the aim of the current study was to reveal the epigenetic mechanism of hepatic transcriptional reprogramming changes based on the integration analysis of RNA-seq and H3K27ac labeled CUT&Tag. Results showed that lipid contents increased gradually with the embryonic age (E) 11, E15, and E19 based on morphological analysis of Hematoxylin-eosin and Oil Red O staining as well as total triglyceride and cholesterol detection. The hepatic protein level of SREBP-1c was higher in E19 when compared with that in E11 and E15, while H3K27ac and H3K4me2 levels declined from E11 to E19. Differential expression genes (DEGs) among these 3 embryonic ages were determined by transcriptome analysis. A total of 107 and 46 genes were gradually upregulated and downregulated respectively with the embryonic age. Meanwhile, differential H3K27ac occupancy in chromatin was investigated. But the integration analysis of RNA-seq and CUT&Tag data showed that the overlap genes were less between DEGs and target genes of differential peaks in the promoter regions. Further, some KEGG pathways enriched from target genes of typical enhancer were overlapped with those from DEGs in transcriptome analysis such as insulin, FoxO, MAPK signaling pathways which were related to lipid metabolism. DNA motif analysis identify 8 and 10 transcription factors (TFs) based on up and down differential peaks individually among E11, E15, and E19 stages where 7 TFs were overlapped including COUP-TFII, FOXM1, FOXA1, HNF4A, RXR, ERRA, FOXA2. These results indicated that H3K27ac histone modification is involved in the transcriptional reprogramming regulation during embryonic development, which could recruit TFs binding to mediate differential enhancer activation. Differential activated enhancer impels dynamic transcriptional reprogramming towards lipid metabolism to promote the occurrence of special phenotype of hepatic lipid deposition.

ABSTRACT: Attaining the optimal feed conversion ratio is the unaltered goal for poultry breeding, meat yield is one of the vital reference indexes for that. Folic acid is involved in protein metabolism by acting as a transmitter of one carbon unit, and the detail mechanism for the high-dose folic acid on growth of broiler skeletal muscle is still unclarified. The present study was conducted to investigate the effect and regulatory mechanism of folic acid on deposition and metabolism of protein in broiler breast muscle. A total of 196 one-day-old AA broilers were randomly assigned to 2 treatment groups. The chicks were fed corn-soybean diet with folic acid levels of 1.3 mg/kg (CON) or 13 mg/kg (FA), respectively. The results showed that high dose of folic acid significantly increased the body weight gain, average daily gain, average daily feed intake, and feed conversion ratio of broilers during 1 to 42 d. Compared with control group, folic acid statistically augmented the breast muscle ratio of broilers at 42 d, abdominal fat percentage was also decreased in FA group. Folic acid significantly increased the gene expression of folate receptor (FR) in duodenum and jejunum at 21 d, and its relative expression in jejunum of broilers at 42 d. Furthermore, relative expression of myogenin in broiler breast muscle was upregulated in folic acid group. Folic acid supplementation significantly enhanced the protein expression of phosphorylated serine/threonine kinase (AKT) and ribosomal protein S6 kinase 1 (S6K1) in the breast muscle of broilers at 21 d and 42 d. In conclusion, the results proved that high-dose folic acid activated the AKT/mammalian target of rapamycin (mTOR) pathway and increased the activity of phosphorylation of S6K1, thereby regulating the protein deposition in breast muscle. Meanwhile, the gene expression of the myogenic determinant factor was upregulated by folic acid and then promoted the growth of breast muscle. Consequently, the growth performance, meat production and feeding efficiency were improved of broilers by adding folic acid at 13 mg/kg.

Body phosphorus homeostasis network allows laying hens to adapt to wide range of changes in dietary phosphorus levels. Phytase hydrolyzes phytate rendering phosphorus and reduces the laying hens' requirements for inorganic phosphate rock. Here, we demonstrate that there is no need to keep large safety margins in dietary phosphorus when hens are fed with phytase. Hy-Line Brown laying hens (n = 504) were randomly assigned to 7 treatments (6 replicates of 12 birds). A corn-soybean meal–based diet, with no inorganic phosphate rock, was formulated to contain 0.12% nonphytate phosphorus (nPP), 3.8% calcium, and 2,000 FTU/kg phytase. Inorganic phosphate rock (di-calcium phosphate) was supplemented into the basal diet to create 6 other diets containing 0.17, 0.22, 0.27, 0.32, 0.37, and 0.42% nPP. Levels of calcium carbonate and zeolite powder were adjusted to make sure all the 7 experimental diets contained the same nutrition levels (including calcium and phytase) except nPP. The diets were subjected to laying hens from 29 to 40 wk of age. As a result, when supplemented with 2,000 FTU/kg phytase, extra supplementation of inorganic phosphate rock had no effects (P > 0.05) on serum phosphorus levels, serum calcium levels, laying performance (laying rate, egg weight, feed intake, feed-to-egg ratio, and unqualified egg rate), egg quality (shell thickness, shell strength, albumen height, yolk color, and Haugh unit), and tibia quality parameters (breaking strength and ash, calcium, and phosphorus contents). Extra supplementation of inorganic phosphate rock linearly increased (P < 0.01) fecal phosphorus excretion and linearly decreased (P = 0.032) the apparent metabolizability of dietary phosphorus. While serum hormones and intestine gene expressions were varied within treatments, no consistent changes were found. In conclusion, the supplementation of inorganic phosphate rock (provided 0.05–0.30% extra nPP) to phytase-containing basal diets (2,000 FTU/kg; nPP = 0.12%) provided limited benefits to egg production performance in laying hens from 29 to 40 wk of age. Further investigating the body phosphorus homeostasis would help to understand the nutritional and physiological reasonability of formulating low-phosphorus diets in the laying hen industry.

The liver is the main site of de novo lipogenesis in poultry, and hepatic lipid metabolism disorder will lead to excessive abdominal fat deposition or fatty liver disease, finally causing huge economic loss. The present study was conducted to investigate developmental changes in hepatic lipid metabolism of chicks from embryonic periods to the first week after hatching. Liver samples were collected from embryonic day 11 (E11) to the age of day 7 posthatch (D7) for lipid metabolism analysis. Hematoxylin–eosin and Oil Red O staining analysis showed that hepatic lipids increased gradually during embryonic period and declined posthatch; The sum of hepatic triglycerides and cholesterol reached the peak at E19 and D1 by ELISA analysis (P < 0.05). Acetyl-CoA carboxylase, fatty acid synthase, and acyl-CoA desaturase 1 mRNA expression in the liver were higher from E17 to D1 with the peak at E19 when compared with those at E13 and E15 (P < 0.05). Hepatic elongase of very long-chain fatty acids 6 and microsomal triglyceride transfer protein mRNA abundance were lower during embryonic periods but reached relative higher level after hatching (P < 0.05). On the contrary, hepatic carbohydrate response element binding protein (ChREBP), carnitine palmitoyltransferase 1, and peroxisome proliferators–activated receptor α expression were higher during embryonic periods but decreased posthatch (P < 0.05). The mRNA abundance of sterol-regulatory element binding protein 1c was the lowest at E13 and E15, then increased gradually from E17 to D1, while decreased from D3 to D7 little by little (P < 0.05). In summary, hepatic lipogenesis genes have different expression patterns during the embryonic periods and the first week of posthatch, which might be activated by ChREBP during embryonic periods; fatty acid oxidation was enhanced around the hatched day but declined posthatch. These findings will broaden the understanding of physiological characteristics and dynamic pattern about hepatic lipid metabolism in chicks.

The pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor (TNF)-α mediate inflammation, which is a protective response by body to ensure removal of detrimental stimuli, as well as a healing process for repairing damaged tissue. The overproduction of pro-inflammatory cytokines can induce autoimmune diseases and can be fatal. The aim of this study was to investigate epigenetic mechanisms in the regulation of pro-inflammatory cytokines expression after lipopolysaccharide (LPS) stimulation of broiler peripheral blood mononuclear cells (PBMC). Gene expression, promoter DNA methylation, and chromatin accessibility of pro-inflammatory cytokines in untreated and LPS-treated PBMC were compared. The expression of epigenetic enzymes DNA methyltransferase (DNMT) 1, histone deacetylase (HDAC), and histone acetylase (HAT) were measured after LPS stimulation. The results showed the activated gene expression of proinflammatory cytokines in broiler PBMC stimulated 3 h by LPS. The demethylation of IL-6 gene -- 302 and -264 cytosine-guanine (CpG) sites, as well as TNF- α gene -371 CpG site, occurred after LPS treatment (P < 0.05), whereas the methylaiton pattern in the IL-1β gene promoter region was not affected. Otherwise, LPS stimulation relaxed the chromatin structure at IL-1β and IL-6 promoter (P < 0.05). The lower expression of DNMT1 and HDAC2, and higher expression of p300- CBP-associated factor and tat-interaction protein-60, were detected in response to LPS (P < 0.05). Our data indicated that after LPS stimulation for 3 h, IL-1β and IL-6 promoter are remodeled into an accessible structure, and the IL-6 and TNF-α promoter are demethylated at special sites, which possible impact the mRNA expression of pro-inflammatory cytokines. [ABSTRACT FROM AUTHOR]