Effect of High Energy Low Protein Diet on Lipid Metabolism and Inflammation in the Liver and Abdominal Adipose Tissue of Laying Hens.

The aim of this study was to evaluate the effects of a high-energy low-protein (HELP) diet on lipid metabolism and inflammation in the liver and abdominal adipose tissue (AAT) of laying hens. A total of 200 Roman laying hens (120 days old) were randomly divided into two experimental groups: negative control group (NC group) and HELP group, with 100 hens per group. The chickens in the NC group were fed with a basic diet, whereas those in the HELP group were given a HELP diet. Blood, liver, and AAT samples were collected from 20 chickens per group at each experimental time point (30, 60, and 90 d). The morphological and histological changes in the liver and AAT were observed, and the level of serum biochemical indicators and the relative expression abundance of key related genes were determined. The results showed that on day 90, the chickens in the HELP group developed hepatic steatosis and inflammation. However, the diameter of the adipocytes of AAT in the HELP group was significantly larger than that of the NC group. Furthermore, the results showed that the extension of the feeding time significantly increased the lipid contents, lipid deposition, inflammatory parameters, and peroxide levels in the HELP group compared with the NC group, whereas the antioxidant parameters decreased significantly. The mRNA expression levels of genes related to lipid synthesis such as fatty acid synthase (FASN), stearoyl-coA desaturase (SCD), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor gamma (PPARγ) increased significantly in the liver and AAT of the HELP group, whereas genes related to lipid catabolism decreased significantly in the liver. In addition, the expression of genes related to lipid transport and adipokine synthesis decreased significantly in the AAT, whereas in the HELP group, the expression levels of pro-inflammatory parameters such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1ß) increased significantly in the liver and AAT. Conversely, the expression level of the anti-inflammatory parameter interleukin-10 (IL-10) decreased significantly in the liver. The results indicated that the HELP diet induced lipid peroxidation and inflammation in the liver and AAT of the laying hens. Hence, these results suggest that chicken AAT may be involved in the development of fatty liver.

RNA-Seq Analysis Reveals the Molecular Mechanisms Regulating the Development of Different Adipose Tissues in Broiler Chicks.

In an effort to enhance growth rates, chicken breeders have undertaken intensive genetic selection. In the selection process, the primary aim is to accelerate growth, inadvertently leading to new chicken breeds having an increased capacity for rapid adipose tissue accumulation. However, little is known about the relationship between changes in gene expression and adipose tissue accumulation and deposition in chickens. Therefore, in this study, RNA-seq analysis was utilized, and transcriptome data were obtained from the abdominal fat, thoracic subcutaneous fat, and clavicular fat on day 1 (d1), day 4, day 7, day 11, and day 15 to reveal the molecular mechanisms regulating the development and deposition of different adipose tissues in broiler chicks. The results showed that the key period for adipocyte differentiation and proliferation was between d4 and d7 (abdominal fat development) and between d1 and d4 (chest subcutaneous fat and clavicular fat). In addition, candidate genes such as MYOG, S100A9, CIDEC, THRSP, CXCL13, and NMU related to adipose tissue growth and development were identified. Further, genes (HOXC9, AGT, TMEM182, ANGPTL3, CRP, and DSG2) associated with the distribution of adipose tissue were identified, and genes (MN1, ANK2, and CAP2) related to adipose tissue growth were also identified. Taken together, the results from this study provide the basis for future studies on the mechanisms regulating adipose tissue development in chickens. Further, the candidate genes identified could be used in the selection process.

Potential therapeutic effects of milk-derived exosomes on intestinal diseases.

Exosomes are extracellular vesicles with the diameter of 30 ~ 150 nm, and are widely involved in intercellular communication, disease diagnosis and drug delivery carriers for targeted disease therapy. Therapeutic application of exosomes as drug carriers is limited due to the lack of sources and methods for obtaining adequate exosomes. Milk contains abundant exosomes, several studies have shown that milk-derived exosomes play crucial roles in preventing and treating intestinal diseases. In this review, we summarized the biogenesis, secretion and structure, current novel methods used for the extraction and identification of exosomes, as well as discussed the role of milk-derived exosomes in treating intestinal diseases, such as inflammatory bowel disease, necrotizing enterocolitis, colorectal cancer, and intestinal ischemia and reperfusion injury by regulating intestinal immune homeostasis, restoring gut microbiota composition and improving intestinal structure and integrity, alleviating conditions such as oxidative stress, cell apoptosis and inflammation, and reducing mitochondrial reactive oxygen species (ROS) and lysosome accumulation in both humans and animals. In addition, we discussed future prospects for the standardization of milk exosome production platform to obtain higher concentration and purity, and complete exosomes derived from milk. Several in vivo clinical studies are needed to establish milk-derived exosomes as an effective and efficient drug delivery system, and promote its application in the treatment of various diseases in both humans and animals.

High expression of miR-22-3p in chicken hierarchical follicles promotes granulosa cell proliferation, steroidogenesis, and lipid metabolism via PTEN/PI3K/Akt/mTOR signaling pathway.

MicroRNAs (miRNAs) are a class of RNA macromolecules that play regulatory roles in follicle development by inhibiting protein translation through binding to the 3'UTR of its target genes. Granulosa cell (GC) proliferation, steroidogenesis, and lipid metabolism have indispensable effect during folliculogenesis. In this study, we found that miR-22-3p was highly expressed in the hierarchical follicles of the chickens, which indicated that it may be involved in follicle development. The results obtained suggested that miR-22-3p promoted proliferation, hormone secretion (progesterone and estrogen), and the content of lipid droplets (LDs) in the chicken primary GC. The results from the bioinformatics analysis, luciferase reporter assay, qRT-PCR, and Western blotting, confirmed that PTEN was directly targeted to miR-22-3p. Subsequently, it was revealed that PTEN inhibited proliferation, hormone secretion, and the content of LDs in GC. Therefore, this study showed that miR-22-3p could activate PI3K/Akt/mTOR pathway via targeting PTEN. Taken together, the findings from this study indicated that miR-22-3p was highly expressed in the hierarchical follicles of chickens, which promotes GC proliferation, steroidogenesis, and lipid metabolism by repressing PTEN to activate PI3K/AKT/mTOR pathway.

Analysis of the Selection Signal of the Tibetan Black Chicken Genome Based on Whole-Genome Sequencing.

BACKGROUND: The Tibetan chicken has adapted well to high altitudes genetically after its long-term habitation in the plateau. In this study, we analyzed the selection signal of Tibetan black chickens (TBCs) and discovered genes associated with the characteristics of germplasm. METHODS: Whole-genome sequencing (WGS) was used to identify the single-nucleotide polymorphism (SNP) markers and genetic structures in the genome of Tibetan black chickens. Further, we performed a comparative population genomics analysis between the genomic data obtained in this present study and the genomic data for five wild red jungle fowls (RJFs) accessed from the NCBI database (GenBank accession number PRJNA241474). Thereafter, the Fst and Pi selections were used to identify genes under positive selection in the Tibetan black chicken genome. RESULTS: A total of 9,490,690 SNPs were identified in the Tibetan black chickens. In addition, the results from the gene ontology (GO) analysis showed that 732 genes of TBCs were enriched in a total of 210 GO terms with specific molecular functions such as regulation of cellular catabolic process, the MAPK signaling pathway, regulation of ion transport, growth, morphogenesis and lung alveolus development which may provide a better mechanism to facilitate oxygen transport and utilization in TBCs. Moreover, the results from the KEGG analysis showed that 732 genes of the TBCs were significantly enriched in the calcium signaling pathway, circadian entrainment (ADCY1, GNG7 and PER3), oxytocin signaling pathway and pathways of multiple neurodegeneration diseases. In addition, the CD86 antigen (CD86) was identified as a gene associated with the immune response in chickens. It was also revealed that genes such as TRIT1, HPCAL4, NT5C1A and HEYL were discovered under selection in Tibetan black chickens on chromosome 23. These genes may be related to the local adaptive characteristics of Tibetan black chickens, for instance, NT5C1A and HEYL may be involved in the high-altitude adaption of oxygen delivery in Tibetan black chickens. CONCLUSIONS: In summary, we found that selection mainly affects the disease resistance and cold acclimatization of Tibetan black chickens. Hence, these results may provide important genetic information for the evolution and breeding of Tibetan black chickens.

Remodeling the hepatic fibrotic microenvironment with emerging nanotherapeutics: a comprehensive review.

Liver fibrosis could be the last hope for treating liver cancer and remodeling of the hepatic microenvironment has emerged as a strategy to promote the ablation of liver fibrosis. In recent years, especially with the rapid development of nanomedicine, hepatic microenvironment therapy has been widely researched in studies concerning liver cancer and fibrosis. In this comprehensive review, we summarized recent advances in nano therapy-based remodeling of the hepatic microenvironment. Firstly, we discussed novel strategies for regulatory immune suppression caused by capillarization of liver sinusoidal endothelial cells (LSECs) and macrophage polarization. Furthermore, metabolic reprogramming and extracellular matrix (ECM) deposition are caused by the activation of hepatic stellate cells (HSCs). In addition, recent advances in ROS, hypoxia, and impaired vascular remodeling in the hepatic fibrotic microenvironment due to ECM deposition have also been summarized. Finally, emerging nanotherapeutic approaches based on correlated signals were discussed in this review. We have proposed novel strategies such as engineered nanotherapeutics targeting antigen-presenting cells (APCs) or direct targeting T cells in liver fibrotic immunotherapy to be used in preventing liver fibrosis. In summary, this comprehensive review illustrated the opportunities in drug targeting and nanomedicine, and the current challenges to be addressed.

miR-128-3p regulates chicken granulosa cell function via 14-3-3ß/FoxO and PPAR-γ/LPL signaling pathways.

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3'UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3ß (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3ß/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3ß/FoxO and PPAR-γ/LPL signaling pathways.

Effects of Dietary Ferulic Acid on Intestinal Health and Ileal Microbiota of Tianfu Broilers Challenged with Lipopolysaccharide.

Lipopolysaccharide (LPS) has been considered the primary agent to establish animal models of inflammation, immunological stress, and organ injury. Previous studies have demonstrated that LPS impaired gastrointestinal development and disrupted intestinal microbial composition and metabolism. Ferulic acid (FA) isolated from multiple plants exhibits multiple biological activities. This study investigated whether FA ameliorated intestinal function and microflora in LPS-challenged Tianfu broilers. The results showed that LPS challenge impaired intestinal function, as evidenced by decreased antioxidant functions (p < 0.05), disrupted morphological structure (p < 0.05), and increased intestinal permeability (p < 0.05); however, these adverse effects were improved by FA supplementation. Additionally, FA supplementation preserved sIgA levels (p < 0.05), increased mRNA expression levels of CLDN and ZO-1 (p < 0.05), and enhanced epithelial proliferation (p < 0.05) in the ileal mucosa in LPS-challenged chickens. Moreover, FA supplementation rectified the ileal microflora disturbances in the LPS-challenged broilers. The results demonstrate that dietary FA supplementation decreased LPS-induced intestinal damage by enhancing antioxidant capacity and maintaining intestinal integrity. Furthermore, FA supplementation protects intestinal tight junctions (TJs), elevates secretory immunoglobulin A (sIgA) levels, and modulates ileal microflora composition in LPS-challenged broilers.

Steatosis in metabolic diseases: A focus on lipolysis and lipophagy.

Fatty acids (FAs), as part of lipids, are involved in cell membrane composition, cellular energy storage, and cell signaling. FAs can also be toxic when their concentrations inside and/or outside the cell exceed physiological levels, which is called "lipotoxicity", and steatosis is a form of lipotoxity. To facilitate the storage of large quantities of FAs in cells, they undergo a process called lipolysis or lipophagy. This review focuses on the effects of lipolytic enzymes including cytoplasmic "neutral" lipolysis, lysosomal "acid" lipolysis, and lipophagy. Moreover, the impact of related lipolytic enzymes on lipid metabolism homeostasis and energy conservation, as well as their role in lipid-related metabolic diseases. In addition, we describe how they affect lipid metabolism homeostasis and energy conservation in lipid-related metabolic diseases with a focus on hepatic steatosis and cancer and the pathogenesis and therapeutic targets of AMPK/SIRTs/FOXOs, PI3K/Akt, PPARs/PGC-1α, MAPK/ERK1/2, TLR4/NF-κB, AMPK/mTOR/TFEB, Wnt/ß-catenin through immune inflammation, oxidative stress and autophagy-related pathways. As well as the current application of lipolytic enzyme inhibitors (especially Monoacylglycerol lipase (MGL) inhibitors) to provide new strategies for future exploration of metabolic programming in metabolic diseases.

MiR-34a-5p promotes autophagy and apoptosis of ovarian granulosa cells via the Hippo-YAP signaling pathway by targeting LEF1 in chicken.

Follicular atresia is a natural physiological phenomenon in poultry reproduction. It is well known that follicular atresia is caused by both autophagy and apoptosis of granulosa cells. In current experiment, we evaluated the function of miR-34a-5p on autophagy and apoptosis in chicken follicular atresia. First, the follicular atresia model of chicken was successfully constructed by subcutaneous injection of tamoxifen (TMX), and found the expression of miR-34a-5p in the atresia follicles obviously increased. Then, we confirmed that miR-34a-5p accelerates autophagy and apoptosis of chicken granulose cells in vitro, and miR-34a-5p could induce apoptosis by mediating autophagy. Mechanistically, lymphoid enhancer binding factor 1 (LEF1) was deemed as a target gene for miR-34a-5p. On the contrary, LEF1 overexpression attenuated the autophagy and apoptosis of chicken granular cells. In addition, it was confirmed that the miR-34a-5p/LEF1 axis plays a regulatory role in chicken granulosa cells by mediating the Hippo-YAP signaling pathway. Taken together, this study demonstrated that miR-34a-5p contributes to autophagy and apoptosis of chicken follicular granulosa cells by targeting LEF1 to mediate the Hippo-YAP signaling pathway.

Study on the role of heat shock protein 90 (HSP90) gene in chicken preadipocytes proliferation and differentiation.

In this study, we examined the effects of Heat Shock Protein 90 (HSP90) on adipocyte proliferation and differentiation in chickens. To achieve this, we constructed RNA interference (RNAi) vectors to target HSP90 and transfected the vectors into primary adipocytes. After transfection, oil red O staining was performed to determine the status of triglyceride accumulation in the cells, whereas the CCK-8 cell kit and 5-Ethynyl-2'-Deoxyuridine (EdU) assays were used to determine cell proliferation. Thereafter, the mRNA and protein expression levels of PPARγ, FAS, SREBP-1c, and HSP90 were determined, and the results showed that after the interference of HSP90, the mRNA and protein expression levels of HSP90 in the chicken adipocytes decreased significantly compared to the control and blank groups (p < 0.05). The decreased mRNA and protein expression of PPARγ, FAS, and SREBP-1c was related to adipocyte differentiation (p < 0.05). However, HSP90 interference had no effect on adipocyte proliferation (p > 0.05). Taken together, the results of this study showed that HSP90 influenced the expression of PPARγ and adipose-differentiation-related genes, thereby regulating triglyceride accumulation and adipocyte differentiation in chickens.

Circadian miR-218-5p targets gene CA2 to regulate uterine carbonic anhydrase activity during egg shell calcification.

MicroRNAs (miRNAs) are involved in regulating the circadian clock. In our previous work, miR-218-5p was found to be a circadian miRNA in the chicken uterus, but its role in the eggshell formation process was not clear. In the present study, we found that the expression levels of miR-218-5p and two 2 predicted target genes carbonic anhydrase 2 (CA2) and neuronal PAS domain protein 2 (NPAS2) were oscillated in the chicken uterus. The results of dual-luciferase reporter gene assays in the present study demonstrated that miR-218-5p directly targeted the 3' untranslated regions of CA2 and NPAS2. miR-218-5p showed an opposite expression profile to CA2 within a 24 h cycle in the chicken uterus. Moreover, over-expression of miR-218-5p reduced the mRNA and protein expression of CA2, while miR-218-5p knockdown increased CA2 mRNA and protein expression. Overexpression of CA2 also significantly increased the activity of carbonic anhydrase Ⅱ (P < 0.05), whereas knockdown of CA2 decreased the activity of carbonic anhydrase Ⅱ. miR-218-5p influenced carbonic anhydrase activity via regulating the expression of CA2. These results demonstrated that clock-controlled miR-218-5p regulates carbonic anhydrase activity in the chicken uterus by targeting CA2 during eggshell formation.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-ß pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-ß, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1ß, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-ß pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Therapeutic application of quercetin in aging-related diseases: SIRT1 as a potential mechanism.

Quercetin, a naturally non-toxic flavonoid within the safe dose range with antioxidant, anti-apoptotic and anti-inflammatory properties, plays an important role in the treatment of aging-related diseases. Sirtuin 1 (SIRT1), a member of NAD+-dependent deacetylase enzyme family, is extensively explored as a potential therapeutic target for attenuating aging-induced disorders. SIRT1 possess beneficial effects against aging-related diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Depression, Osteoporosis, Myocardial ischemia (M/I) and reperfusion (MI/R), Atherosclerosis (AS), and Diabetes. Previous studies have reported that aging increases tissue susceptibility, whereas, SIRT1 regulates cellular senescence and multiple aging-related cellular processes, including SIRT1/Keap1/Nrf2/HO-1 and SIRTI/PI3K/Akt/GSK-3ß mediated oxidative stress, SIRT1/NF-κB and SIRT1/NLRP3 regulated inflammatory response, SIRT1/PGC1α/eIF2α/ATF4/CHOP and SIRT1/PKD1/CREB controlled phosphorylation, SIRT1-PINK1-Parkin mediated mitochondrial damage, SIRT1/FoxO mediated autophagy, and SIRT1/FoxG1/CREB/BDNF/Trkß-catenin mediated neuroprotective effects. In this review, we summarized the role of SIRT1 in the improvement of the attenuation effect of quercetin on aging-related diseases and the relationship between relevant signaling pathways regulated by SIRT1. Moreover, the functional regulation of quercetin in aging-related markers such as oxidative stress, inflammatory response, mitochondrial function, autophagy and apoptosis through SIRT1 was discussed. Finally, the prospects of an extracellular vesicles (EVs) as quercetin loading and delivery, and SIRT1-mediated EVs as signal carriers for treating aging-related diseases, as well as discussed the ferroptosis alleviation effects of quercetin to protect against aging-related disease via activating SIRT1. Generally, SIRT1 may serve as a promising therapeutic target in the treatment of aging-related diseases via inhibiting oxidative stress, reducing inflammatory responses, and restoring mitochondrial dysfunction.

Gga-miR-146b-3p promotes apoptosis and attenuate autophagy by targeting AKT1 in chicken granulosa cells.

The normal development of follicles determines the reproductive performance of females. Granulosa cells (GC) play crucial roles in follicular maturation. Numerous studies have shown that miRNAs are involved in the regulation of GC. According to our previous sequencing data, gga-miR-146b-3p was differentially expressed in normal and atretic chicken follicles. In this study, we verified that gga-miR-146b-3p attenuated proliferation and autophagy but promoted apoptosis in chicken GC. Threonine kinase1 (AKT1), a key member of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, was predicted to be a target gene of gga-miR-146b-3p via bioinformatic analysis. Dual-luciferase reporter gene assays were used to determine target relationships. Moreover, knockout of AKT1 decelerated proliferation and autophagy while accelerating the apoptosis of GC. However, overexpression of AKT1 reversed these results. In summary, our results demonstrated that gga-miR-146b-3p repressed the proliferation and autophagy of chicken GC while up-regulating apoptosis by targeting AKT1 through the PI3K/AKT signaling pathway. These findings may provide great insights for further exploration of the molecular regulation of gga-miR-146b-3p and AKT1 on the functions of GC during folliculogenesis.

Synergy of Dietary Quercetin and Vitamin E Improves Cecal Microbiota and Its Metabolite Profile in Aged Breeder Hens.

In the present study, the synergistic effects of quercetin (Q) and vitamin E (E) on cecal microbiota composition and function, as well as the microbial metabolic profile in aged breeder hens were investigated. A total of 400 (65 weeks old) Tianfu breeder hens were randomly allotted to four experimental groups (four replicates per group). The birds were fed diets containing quercetin at 0.4 g/kg, vitamin E (0.2 g/kg), quercetin and vitamin E (QE; 0.4 g/kg and 0.2 g/kg), and a basal diet for a period of 10 wks. After the 10 week experimental period, the cecal contents of 8 aged breeder hens per group were sampled aseptically and subjected to high-throughput 16S rRNA gene sequencing and untargeted metabolomic analysis. The results showed that the relative abundances of phyla Bacteroidota, Firmicutes, and Actinobacteriota were the most prominent among all the dietary groups. Compared to the control group, the relative abundance of the families Bifidobacteriaceae, Lachnospiraceae, Tannerellaceae, Mathonobacteriaceae, Barnesiellaceae, and Prevotellaceae were enriched in the QE group; and Bacteroidaceae, Desulfovibrionaceae, Peptotostretococcaceae, and Fusobacteriaceae were enriched in the Q group, whereas those of Lactobacillaceae, Veillonellaceae, Ruminococcaceae, Akkermansiaceae, and Rikenellaceae were enriched in the E group compared to the control group. Untargeted metabolomics analyses revealed that Q, E, and QE modified the abundance of several metabolites in prominent pathways including ubiquinone and other terpenoid-quinone biosynthesis, regulation of actin cytoskeleton, insulin secretion, pancreatic secretion, nicotine addiction, and metabolism of xenobiotics by cytochrome P450. Furthermore, key cecal microbiota, significantly correlated with important metabolites, for example, (S)-equol positively correlated with Alistipes and Chlamydia in E_vs_C, and negatively correlated with Olsenella, Paraprevotella, and Mucispirillum but, a contrary trend was observed with Parabacteroides in QE_vs_C. This study establishes that the synergy of quercetin and vitamin E alters the cecal microbial composition and metabolite profile in aged breeder hens, which lays a foundation for chicken improvement programs.

Integrated Proteomic and Metabolomic Analyses of Chicken Ovary Revealed the Crucial Role of Lipoprotein Lipase on Lipid Metabolism and Steroidogenesis During Sexual Maturity.

During sexual maturation and ovulatory cycle in chickens, ovaries undergo dynamic morphological and functional changes. The aim of this study was to evaluate the integrated proteome and metabolome analyses of chicken ovaries to characterize the changes in protein and metabolite profiles during sexual maturity. The ovary of Rohman layers before (125 days of age) and after (139 days of age) sexual maturation were collected for proteome and metabolome sequencing. The results showed that a total of 680 differentially expressed proteins (DEPs) and 1,046 differential metabolites (DMs) were identified in the chicken ovary during sexual maturity. Among the DEPs, 595 proteins were up-regulated and 85 were down-regulated, whereas 519 metabolites were up-regulated and 527 were down-regulated. KEGG pathway enrichment analysis showed that DEPs were significantly enriched in glycerolipid metabolism, calcium signaling pathway, folate biosynthesis, fat digestion and absorption, NF-kB signaling pathway, and PPAR signaling pathway. However, DMs were significantly enriched in the metabolism pathways, PPAR signalling pathway, glycerolipid metabolism, ferroptosis, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids. The results of the integrated analyses of DEPs and DMs revealed that the PPAR signaling pathway and glycerolipid metabolism were the most significantly enriched pathways. Among the identified DEPs, lipoprotein lipase (LPL) was upregulated in sexually mature chicken ovaries and was significantly enriched in the glycerolipid metabolism pathway, which may partially explain the possible reasons for steroidogenesis and lipid reserves responsible for oocyte maturation and ovarian follicle development during sexual maturity in chickens. The results further revealed that LPL silencing decreased the content of lipid droplets (LDs), as well as the mRNA expression of lipid metabolism-related genes including; sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase (FASN); and steroidogenesis-related genes such as; cytochrome P450 11A1 (CYP11A1) and steroidogenic acute regulatory (StAR). The present study revealed that upregulation of LPL in the chicken ovary during sexual maturity promotes granulosa cell (GC) lipid metabolism and steroidogenesis. These findings provide a theoretical support for further studies to elucidate the mechanism of lipid metabolism to regulate the function of avian GCs during sexual maturity in chickens.

Supplementation of Dietary Quercetin and Vitamin E Promotes the Intestinal Structure and Immune Barrier Integrity in Aged Breeder Hens.

In aged animals, the physiological functions of the gastrointestinal tract (GIT) are reduced. Dietary intervention is necessary to re-activate GIT functions. The objective of this study was to investigate the impacts of dietary combination of quercetin (Q) and vitamin E (VE) on the intestinal structure and barrier integrity in aged breeder chickens. A sum of 400 (65-wks-old) Tianfu breeder hens were randomly allotted into four (4) groups with four (4) replicates, and fed with basal diet; basal diet supplemented with 0.4g/kg of Q; basal diet supplemented with 0.2g/kg of VE; and basal diet supplemented with the combination of Q (0.4 g/kg) and VE (0.2 g/kg) for 14 weeks. At the end of the 14th week, serum and gut segments were collected from eight hens per group for analyses. The results showed that Q+VE exerted synergistic effects on intestinal morphology by promoting villi height and crypt depth (P < 0.05), as well as mitigated the intestinal inflammatory damage of the aged hens, but decreased the concentration of serum D-lactate and diamine oxidase; and increased the levels of secretory immunoglobulin A (sIgA) and Mucin-2 mRNA (P < 0.05). Furthermore, the mRNA expression of intestinal tight junction proteins including occludin, ZO1, and claudin-1 was increased by Q+VE (P < 0.05). Moreover, Q+VE decreased the mRNA expression of the pro-inflammatory genes (TNF-α, IL-6, and IL-1ß), and increased the expression of anti-inflammatory genes (IL-10 and IL-4) (P < 0.05). These results were consistent with the mRNA expression of Bax and Bcl-2. In addition, Q+VE protected the small intestinal tract from oxidative damage by increasing the levels of superoxide dismutase, total antioxidant capacity, glutathione peroxidase, catalase (P < 0.05), and the mRNA expression of SOD1 and GPx-2. However, Q+VE decreased malondialdehyde levels in the intestine compared to the control (P < 0.05). These results indicated that dietary Q+VE improved intestinal function in aged breeder hens, by protecting the intestinal structure and integrity. Therefore, Q+VE could act as an anti-aging agent to elevate the physiological functions of the small intestine in chickens.