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.

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.

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.

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.

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.

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.

Altitude-adaption of gut microbiota in Tibetan chicken.

Low oxygen levels and extremely cold weather in high-altitude environments requires more energy intake to maintain body temperature in animals. However, little is known about the characteristics of cecal and ileac microbiota in Tibetan chicken and how the high and low altitude environments affect the gut microbiota communities in Tibetan chicken. In the present study, In the present study, Tibetan chickens (Group HA, 3572 m, 578.5 Pa) and their introduced flatland counterparts (Group LA, 580 m, 894.6 Pa) in the cecum and ileum to identify the possible bacterial species that are helpful for their host in environmental adaption. High-throughput sequencing was used to sequence the V3 to V4 hypervariable regions of the bacterial 16S rRNA gene. By comparing the gut microbial diversity of HA chicken with that of LA, the results indicated that the microbial diversity of the cecum and ileum in group HA was significantly lower (P < 0.05) than those in group LA. The cecum microbiome maintained higher population diversity and richness than the ileum (P < 0.05). Four phyla Firmicutes, Bacterioidetes, Actinobacteria, and Proteobacteria were dominant in two groups. Interestingly, there were significant differences in abundance ratio among the four groups (P < 0.05). The predominant bacteria in HA and LA ileum belong to Proteobacteria and Firmicutes, whereas in cecum, Bacterioidetes and Actinobacteria were predominant in both groups (P < 0.05). Correlation analysis showed that Sporosarcina, Enterococcus, and Lactococcus were strongly related to air pressure, and Peptoclostridium and Ruminococcaceae_UCG-014 are related to altitude and gut microbiota of LA group was influenced by altitude, while HA group affected by air pressure. Meanwhile, the Ruminococcus-torques-group was negatively correlated with the relative abundance of Paenibacillus, and positive correlated with those of other microorganisms. Furthermore, HA has higher abundance of microbiota involved in energy and glycan biosynthesis metabolism pathway, while LA has higher abundance of microbiota involved in membrane transport, signal transduction, and xenobiotics biodegradation and metabolism. Generally, our results suggested that the composition and diversity of gut microbes changed after Tibetan chickens were introduced to the plain. Tibetan chicken may adapt to new environment via reshaping the gut microbiota. Gut microbes may contribute to the host adaption to high altitude environments by increasing host energy and glycan biosynthesis.

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.

Dietary quercetin and vitamin E supplementation modulates the reproductive performance and antioxidant capacity of aged male breeder chickens.

Aged male chickens experience rapid declines in spermatogenesis, antioxidant capacity, immunity, and hormone synthesis. Vitamin E plays a significant role in reproduction, nervous system function, and disease resistance in animals. Quercetin also exerts many biological effects, such as antioxidant ability, immunostimulation, and protection of spermatozoal plasma membranes. This study evaluated the effects of combining dietary quercetin (Q) and vitamin E (VE) on sperm quality, antioxidant capacity, immunity, and expression of genes related to spermatogenesis, immunity, apoptosis, and inflammation in aged male chickens. A total of 120 Tianfu breeder male chickens (65 wk old) were randomly allotted to 4 treatments with 3 replicates (10 birds each). The birds were fed diets containing Q (0.4g/kg), VE (0.2g/kg), Q+VE (0.4g/kg + 0.2g/kg), and a basal diet for 11 wk. At the end of the experimental period, blood, semen, liver, testes, and spleen samples were collected from 2 birds per replicate. Serum hormones, antioxidant parameters, cytokines, and immunoglobulins were evaluated; and the mRNA expression of genes related to spermatogenesis, apoptosis, and inflammation are determined in the testes and liver tissues. The results showed that the combination quercetin and vitamin E significantly promoted the sperm count and motility, as well as elevated the levels of testosterone, follicle-stimulating hormone, and luteinizing hormone, antioxidant enzymes (Superoxide dismutase, Glutathione, and Total antioxidant capacity), and serum immunoglobulins (IgA and IgM) in the aged male chickens; also Q+VE showed protective effects on the liver against injury. In addition, Q+VE significantly increased the expression of genes related to spermatogenesis (AR, pgk2, Cyclin A1, and Cyclin A2), immunity (IFN-γ and IL-2), and anti-inflammatory cytokines (IL-10) (P < 0.05), whereas the expression of proinflammatory cytokines (IL-1ß and IL-6) was decreased (P < 0.05). Taken together, these data indicate that the combination of quercetin and vitamin E improved reproductive characteristics such as spermatogenesis, sperm quality, and hormone regulation, as well as promoted antioxidant defense, hepatoprotective capacity, and immune response in aged male chickens without any detrimental effects.

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.

Synergy Between Dietary Quercetin and Vitamin E Supplementation in Aged Hen's Diet Improves Hatching Traits, Embryo Quality, and Antioxidant Capacity of Chicks Hatched From Eggs Subjected to Prolonged Storage.

The current study aims to investigate the effects of the synergy between quercetin and vitamin E in aged hen's diet on hatchability and antioxidant levels of the embryo and newly hatched chicks from prolonged storage eggs. A total of 400 breeder laying hens of 65 weeks of age were selected and randomly divided into 4 groups. Birds were fed a basal diet alone (Control), and basal diets supplemented with quercetin (Q) (0.4 g/kg) and vitamin E (VE) (0.2 g/kg) alone and their combination (0.4 g/kg Q + 0.2 g/kg VE) for 14 weeks, respectively, to determine their effects on yolk antioxidant status, fertility, embryonic mortality, hatchability, antioxidant status of embryonic tissues, as well as the antioxidant status of the newly hatched chicks. The results showed that the hen's dietary Q + VE increased the yolk weight, as well as increased the antioxidant status of the egg yolk (p < 0.05). Compared with the control group, the supplementation of Q + VE significantly increased the hatchability of set-fertile eggs and decreased early embryonic mortality in eggs stored for 7 and 14 days, respectively (p < 0.05), and also improved the antioxidant capacity of the embryos obtained from eggs stored for 14 days (before incubation) (p < 0.05). Moreover, Q + VE increased the levels of SOD, GSH-Px, T-AOC, T-SOD, and CAT in the liver, heart, and pectoral muscle of the embryo, 1-day-old and 14-day-old chicks (p < 0.05), as well as upregulated the antioxidant related genes (GPx-1, GPx-2, GPx-4, DIO-1, and SOD-1) in the liver of the embryo, 1-day-old and 14-day-old chicks hatched from 14-days storage eggs (p < 0.05). Meanwhile, the MDA levels were decreased by the Q + VE in the embryo and post-hatched chicks (p < 0.05). In conclusion, these findings suggested that maternal dietary Q + VE exerts beneficial synergistic effects on the antioxidant capacity of the egg yolk, embryo, and chicks during prolong egg storage, therefore, Q + VE could be used as a dietary measure to enhance hatchability and chick quality in poultry production.

A transcriptome analysis for 24-hour continuous sampled uterus reveals circadian regulation of the key pathways involved in eggshell formation of chicken.

Circadian timing system controlled the rhythmic events, for example, ovulation and oviposition in chickens. However, how biological clock mediates eggshell formation remains obscure. Here, A 24-h mRNA transcriptome analysis was carried out in the uterus of 18 chickens with similar oviposition time points to identify the rhythmic genes and to reveal critical genes and biological pathways involved in the eggshell biomineralization. JTK_CYCLE analysis and real-time PCR revealed a total of 1,793 genes from the sequencing database with 23,513 genes (FPKM>1) were rhythmic genes regulating the rhythmic system and the expression of typical clock genes Per2, Cry1, Bmal1, Clock, Per3, and Rev-erbß were rhythmically expressed, which suggested that endogenous clock in uterus might control the eggshell mineralization. Time of peak expression of the rhythmic genes was analyzed based on their acrophase. The main phases clustered at the periods from Zeitgeber time 0 (ZT0) to ZT4 (6:00-10:00) and from ZT10 to ZT14 (16:00-20:00). The rhythmic genes were annotated to the following Gene Ontology terms rhythmic process, lyase, ATP binding, cell membrane component. KEGG pathway enrichment analysis revealed the top 15 rhythmic genes were involved in vital biological pathways, including syndecan (1, 2, 3)-mediated signaling, post-translational regulation of adheres junction stability and disassembly, FoxO family signaling, TGF-ß receptor and transport of small molecular pathways. 166 of total 1,235 genes (13.4%) were defined as rhythmic transfer factors (TFs) and they were investigated expression time distribution of cis-elements of circadian clock system D-box, E-box, B-site, and Y-Box within 24 h. Results indicated that rhythmic TFs at each phase are potential drivers of their circadian transcription activities. Compared with the control, the expression abundances of ion transport elements SCNN1G, CA2, SPP1, and ATP1B1 were significantly decreased after the interference of Bmal1 gene in synchronized uterine tubular gland cells. Clock genes changed their expression along with the eggshell formation, indicating that there is circadian clock in the uterus of chicken and it regulates the expression of eggshell formation genes.

Circadian miR-449c-5p regulates uterine Ca2+ transport during eggshell calcification in chickens.

BACKGROUND: miRNAs regulate circadian patterns by modulating the biological clocks of animals. In our previous study, we found that the clock gene exhibited a cosine expression pattern in the fallopian tube of chicken uterus. Clock-controlled miRNAs are present in mammals and Drosophila; however, whether there are clock-controlled miRNAs in the chicken uterus and, if so, how they regulate egg-laying rhythms is unclear. In this study, we selected 18 layer hens with similar ovipositional rhythmicity (each of three birds were sacrificed for study per 4 h throughout 24 h); their transcriptomes were scanned to identify the circadian miRNAs and to explore regulatory mechanisms within the uterus of chickens. RESULTS: We identified six circadian miRNAs that are mainly associated with several biological processes including ion trans-membrane transportation, response to calcium ion, and enrichment of calcium signaling pathways. Verification of the experimental results revealed that miR-449c-5p exhibited a cosine expression pattern in the chicken uterus. Ca2+-transporting ATPase 4 (ATP2B4) in the plasma membrane is the predicted target gene of circadian miR-449c-5p and is highly enriched in the calcium signaling pathway. We speculated that clock-controlled miR-449c-5p regulated Ca2+ transportation during eggshell calcification in the chicken uterus by targeting ATP2B4. ATP2B4 mRNA and protein were rhythmically expressed in the chicken uterus, and dual-luciferase reporter gene assays confirmed that ATP2B4 was directly targeted by miR-449c-5p. The expression of miR-449c-5p showed an opposite trend to that of ATP2B4 within a 24 h cycle in the chicken uterus; it inhibited mRNA and protein expression of ATP2B4 in the uterine tubular gland cells. In addition, overexpression of ATP2B4 significantly decreased intracellular Ca2+ concentration (P < 0.05), while knockdown of ATP2B4 accelerated intracellular Ca2+ concentrations. We found similar results after ATP2B4 knockdown by miR-449c-5p. Taken together, these results indicate that ATP2B4 promotes uterine Ca2+ trans-epithelial transport. CONCLUSIONS: Clock-controlled miR-449c-5p regulates Ca2+ transport in the chicken uterus by targeting ATP2B4 during eggshell calcification.

Synergistic effects of quercetin and vitamin E on egg production, egg quality, and immunity in aging breeder hens.

Laying hens experience a rapid decline in egg production, egg quality, and immunity, usually at the end of the peak laying period. Quercetin, a known flavonoid, exerts biological activities, including phytoestrogenic, immunity, antibiotic, antioxidant, and anti-inflammatory properties. Vitamin E also shows egg production and immunoregulatory potential in animals. This study evaluated the capacity of dietary quercetin, vitamin E, and the combination of both, to promote egg production and egg quality, and to improve the immunity of aging breeder hens. We also elucidated how quercetin and vitamin E combination could synergistically affect egg production, egg quality, and immunity in aging breeder hens. A total of 400 Tianfu broiler breeders at the age of 52 wk were randomly allotted to 4 treatments with 4 replicates, 100 hens per treatment and 25 hens per replicate. They were fed diets containing quercetin at 0.4 g/kg, Vitamin E (200 mg/kg), quercetin and vitamin E (0.4 g/kg and 200 mg/kg), and a basal diet (control) for a period 10 wk. Daily feed intake and egg production rate were recorded, and weekly records were recorded on egg quality tests. At the end of the 10-wk experimental period, blood samples and immune organ (spleen) were collected from 2 birds per replicate, totaling 32 birds. Feed intake, immune organ index, serum cytokines, and immunoglobulins were evaluated, and the mRNA expression of genes related to immunity was determined from the spleen tissue. Generally, the results showed that separately or as a combination, supplemental quercetin and vitamin E significantly improved performance and egg quality (P < 0.05), and significantly increased serum immunoglobulins (IgA, IgM, and IgG) and cytokines (IFN-γ and IL-2) concentrations, as well as promoted immune organ development and index, and promoted the expression of splenic immune-related genes (IL-2 and INF-γ) (P < 0.05), compared with the control. It was confirmed in this study that the combination of quercetin and vitamin E exert synergistic effects on egg production, egg quality, and immune function in aging hens.

Combination of Quercetin and Vitamin E Supplementation Promotes Yolk Precursor Synthesis and Follicle Development in Aging Breeder Hens via Liver-Blood-Ovary Signal Axis.

The fertility of female animals is negatively correlated with increasing chronological age. In aging broiler breeder hens, there is a decline in the functionality of the ovary and liver accompanied by hormonal or endocrine changes, a reduction in antioxidant capacity, and a decrease in folliculogenesis. Therefore, improving the reproductive function in aging breeder hens using dietary strategies is of great concern to the poultry breeder. This study evaluated the capacity of dietary quercetin (Q), vitamin E (VE), and their combination (Q + VE) to promote follicle development and attenuate organ inflammation by improving the antioxidant capacity of aging breeder hens. In this study, 400 broiler breeder hens (Tianfu broilers breeder hens, 435 days old) were allotted into four groups (100 birds each) with four replicates each (25 birds each). They were fed diets containing Q (0.4 g/kg), VE (0.2 g/kg), Q + VE (0.4 g/kg + 0.2 g/kg), and a basal diet for 10 weeks. The results showed that Q + VE improved the organ characteristics (p < 0.05), and also that Q + VE showed protective effects on the liver against injury, as well as increasing the antioxidant capacity of the liver, serum, and ovary (p < 0.05). Furthermore, liver lipid synthesis was increased remarkably, as indicated by the changes in triglyceride levels in hens fed Q + VE (p < 0.05). Levels of E2, FSH, and LH, their receptors, and mRNAs related to yolk precursor synthesis were increased by the Q + VE (p < 0.05). Therefore, the combination of quercetin and vitamin E synergistically promotes and regulates the transportation and exchange of synthetic substances among the liver-blood-ovary alliances to ensure the synchronous development and functional coordination between the liver and ovary in aging breeder hens.

Rearing system influences the testicular development, semen quality and spermatogenic cell apoptosis of layer roosters.

The objective of this study was to compare the testicular development, semen quality, and spermatogenic cell apoptosis of roosters reared in colony, single, and large cages. Rohman parental layers (n = 540) were randomly allocated into cages of rearing system groups (135 males and 405 females). The experimental period was 70 to 210 d of age. We compared testicular development and plasma main reproductive hormones (Follicle-stimulating hormone; Luteinizing hormone; Testosterone; Estrogen2;) from d 70 to 210 of roosters among the three systems. In addition, routine semen quality indexes, apoptosis of testicular spermatogenic cells and sperm apoptosis of breeding roosters under three rearing systems on d 175 and d 210 were evaluated. Roosters during the growing period (from d 70 to 140) have rapid testis growth and increasing main reproductive hormones in plasma. At the peak of sexual maturity (d 210), in colony cage, the females have a positive effect and promote the testis development of males. However, the stocking density in colony cage has no effect on testicular development; compared with the single and large cage. Roosters reared in the natural mating system had better semen quality, particularly in semen volume, density, and viability; the hatching % of fertilized eggs and healthy chicks were higher for the colony than single and large cages. Furthermore, the sperm density was higher for colony than single and large cages, which was related to the apoptosis of spermatogonia and spermatocyte, not the apoptosis of mature sperm. This study provided the basic data for the reproductive performance research of chicken reared in the colony cages.

Adaptive immunity profiling associated with histological subtypes and postoperative survival in gastric cancer.

BACKGROUND: In gastric cancer (GC), abnormal adaptive immunity is correlated with chronic inflammatory disorders and poor prognosis. However, the global study of adaptive immunity involving genes expression is insufficient. METHODS: In this study, we investigated the transcriptional profile of adaptive immunity involving genes in GC from TCGA (The Cancer Genome Atlas). The relevance of adaptive immunity and the clinical features of patients with GC were assessed. Differences in gene expression between each feature and the correlation between gene expression and prognosis were elucidated. RESULTS: According to the expressional profile of adaptive immunity-related genes, 412 patients with GC were grouped into two primary classifications and three secondary classifications. There were no differences in prognosis detected between each subgroup. In the immune subgroups, the distributions of pathological type were obviously different. Additionally, histological types, AJCC (American Joint Committee on Cancer) stage features, grade, tumor stage, aneuploidy score, and fraction genome altered in different subgroups were significantly discrepant. There were 95 differently expressed genes (DEGs) detected between each histological type, which were represented by LAIR1, BTK and LAT2. According to identification of DEGs in the MSTAD (mucinous stomach adenocarcinoma) and SRCC (signet ring cell carcinoma) types, which were relevant to the best and worst prognosis types, respectively, we constructed a model combining seven genes to recognize the MSTAD type (AUC =0.91) and a model combining six genes to recognize the SRCC type (AUC =0.91). Moreover, the expression of FGL1 gene was notably contrasting among the different histological types, and the high-expression of FGL1 was correlated with a poor prognosis. CONCLUSIONS: This study showed that the expressional patterns of adaptive immunity-related genes are closely related to the histological type of GC, and demonstrated that the expression of immune molecules is correlated to the prognosis. Our results are expected to promote immunological therapy for GC.

Hypericin-loaded graphene oxide protects ducks against a novel duck reovirus.

Novel duck reovirus (NDRV) disease is a serious infectious disease for poultry, for which no effective therapy has been established. Therefore, development of novel antivirals against NDRV is urgently needed. In present study, we developed a complex wherein hypericin (HY), which shows broad-spectrum antiviral activity, was loaded onto graphene oxide (GO), which has a high drug-loading capacity and low cytotoxicity. The antiviral activity of the complex (GO/HY) was studied in DF-1 cells and in ducklings infected with the NDRV TH11 strain. GO/HY showed a dose-dependent inhibition of NDRV replication, which may be attributed to direct virus inactivation or inhibition of virus attachment. Western blotting and indirect immunofluorescence assay (IFA) showed markedly suppressed protein expression in GO/HY-treated NDRV-infected DF-1 cells. Moreover, GO/HY prolonged the survival time of the ducklings by reducing pathological lesions caused by the infection and inhibiting viral replication in the liver and lungs. These results suggest that GO/HY has antiviral activity against NDRV both in vitro and in vivo.