| Reproductive performance and gut function of female animals are negatively correlated with increasing chronological age.In aging broiler breeder hens,there is a decline in organ function,yolk precursor synthesis,fertility,laying rate,hatchability,gut microbiota composition and metabolite profile.Nutrition and age are positively correlated,hence,developing nutritional strategies to improve the molecular mechanisms regulating reproductive and physiological performance of aging breeder hens are of great concern to the poultry breeder.Quercetin,a known flavonoid,exerts biological activities,including phytoestrogenic,immunity,antibiotic,antioxidant,and anti-inflammatory properties.Vitamin E also shows antioxidant,egg production,embryonic development,fertility and immunoregulatory potential in animals.Accumulating evidences indicated that quercetin or vitamin E promote reproductive,antioxidant,and physiological functions in breeder hens,however,the mechanisms through which the individual supplements or in combination actively enhance the molecular mechanisms regulating the reproductive,immunity,fertility,hatchability,gut health,and anti-oxidative functions of aged breeder hens have not been established yet.Therefore,the objective of this study was to investigate the effect of dietary quercetin（Q）,vitamin E（VE）,and their combination on the mechanisms regulating reproductive performance and cecal microbiota and metabolite profiles in aging breeder hens.A total of 400 Tianfu broiler breeder hens（52 weeks,with laying rate>50%）were randomly allotted into four（4）groups with four（4）replicates.The chickens were fed a basal diet;basal diet supplemented with 0.4 g/kg quercetin;basal diet supplemented with 0.2 g/kg vitamin E;and basal diet supplemented with the combination of 0.4 g/kg quercetin and 0.2 g/kg vitamin E for 10 weeks.At the end of the 10-week experimental period,tissue samples were collected from 2 birds per replicate,totaling 32 birds for subsequent analyses.The main results are as follows:1.The results showed that separately or as a combination,supplemental quercetin and vitamin E significantly improved egg laying performance and egg quality indices（P<0.05）,and significantly increased serum immunoglobulins（Ig A,Ig M,and Ig G）and cytokines（IFN-γand 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.2.The results also showed that Q+VE improved the organ characteristics（P<0.05）,and also showed protective effects against liver injury,as well as increased 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 the triglyceride levels in hens fed Q+VE（P<0.05）.Levels of E2,FSH,and LH and their receptors,and m RNAs related to yolk precursor synthesis were increased by the Q+VE（P<0.05）.3.Furthermore,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 aging hens,but decreased the concentration of serum D-lactate and diamine oxidase;and increased the levels of secretory immunoglobulin A（s Ig A）and Mucin-2 m RNA（P<0.05）.Furthermore,the m RNA 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 m RNA expression of pro-inflammatory genes,and increased the expression of anti-inflammatory related genes（P<0.05）.In addition,Q+VE protected the small intestinal tract from oxidative damage by increasing the levels of antioxidant enzymes and related genes（P<0.05）.4.16S r RNA gene sequencing and untargeted metabolomic analysis of the cecal contents showed that the relative abundances of phyla Bacteroidota,Firmicutes,and Actinobacteriota were the most prominent among all the groups.Compared to the control group,the relative abundance of the families Bifidobacteriaceae,Lachnospiraceae,Tannerellaceae,Mathonobacteriaceae,Barnesiellaceae,and Prevotellaceae were enriched in the Q+VE 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 VE group compared to the control group.Untargeted metabolomics analyses revealed that Q,VE,and Q+VE 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,e.g.（S）-equol positively correlated with Alistipes and Chlamydia in VE＿vs＿C,and negatively correlated with Olsenella,Paraprevotella,and Mucispirillum however,a contrary trend was observed with Parabacteroides in Q+VE＿vs＿C.5.In addition,the results showed that the hen?s dietary Q+VE increased the yolk characteristics（P<0.05）,as well as significantly increased the hatchability of set-fertile eggs and decreased early embryonic mortality in eggs stored for 7 and 14days,respectively（P<0.05）,and also improved the antioxidant capacity of the embryos obtained from eggs stored for 14 days（before incubation）compared with the control group（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）,and upregulated the antioxidant related genes in the liver of the embryo,1-day-old and 14-day-old chicks hatched from14-days storage eggs（P<0.05）.Taken together,the results indicate that combination of dietary quercetin and vitamin E exert synergistic effects on egg production,egg quality,and immune function in aging hens,as well as promotes and regulates the transport and exchange of synthetic substances between the liver-blood-ovary alliances to ensure synchronous development and functional coordination between the liver and ovary in aging breeder hens.Moreover,the synergy of quercetin and vitamin E improved intestinal function in aging breeder hens,by protecting the intestinal structure and integrity,Q+VE could act as an anti-aging agent to elevate the physiological functions of the small intestine in chickens.In addition,the findings from this study provide valuable information for understanding the mechanisms by which Q+VE promotes productivity in aging breeder hens by altering the gut microbiota and metabolite profiles,providing information on biomarker discovery and validation that facilitate layer breeding and genetic improvement programs.Finally,it is suggested in this study 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. |
