Mechanism Of Eggshell Ultrastructure Formation And Nutritional Modulation In The Late Phase Production Of Laying Hens

Poor eggshell quality in the late laying period is a major threat to the quality and safety of egg products,which brought great economic loss to the poultry industry and egg processing industry.Age-related abnormalities in ultrastructure could be one reasonable explanation for the compromised shell quality in the late laying period.In addition,our previous results indicated that dietary manipulation could enhance eggshell mechanical properties in the late laying period by improving its ultrastructure quality.However,ultrastructural variations of eggshell in the late phase production of laying hens and the underlying mechanism for these age-related changes are still not well understood.Therefore,this study was aimed to explore the mechanism of age-related variations in eggshell ultrastructure and then dietary essential oil supplementation was employed to improve ultrastructure and mechanical properties of eggshell in the late phase of production.The aim of experiment 1 was to evaluate age-associated changes in eggshell quality and ultrastructure and then RNA-sequencing was conducted to investigate the transcriptomic differences in uterus of laying hens in different laying periods(42 vs.72 wk).Results showed that breaking strength and fracture toughness of eggshell declined in aged hen group compared to those in young counterparts,accompanied with ultrastructure variations including the increased thickness of mammillary layer and the decreased incidence of early fusion(P < 0.05).A total of 183 differentially expressed genes(DEGs;125 upregulated and 58 downregulated;FC > 1.5,FDR < 0.05)were identified in uterus of laying hens in the late phase in relative to those at peak production.The DEGs annotated to Gene Ontology terms related to antigen processing and presentation were all downregulated in aged hens(P < 0.05).Besides,some genes encoding matrix proteins such as ovalbumin,versican and glypican 3,which are involved in eggshell biomineralization,were also differentially expressed between groups(P < 0.05).These findings indicated that the altered gene expression of matrix proteins along with the compromised immune function in uterus of late-phase laying hens may conduce to age-related impairments of eggshell ultrastructure and mechanical properties.In experiment 2,a uterine inflammation model induced by lipopolysaccharide(LPS)treatment was established to investigate the effects of uterine inflammation on eggshell biomineralization,ultrastructure and mechanical properties of the resultant eggshell in laying hens.Results showed that LPS injection led to abnormal structures in mammillary layer(like erosion of mammillary caps and increased incidence of type B mammillae),sparse shell member fibers,and weak links between membrane and mammillary knobs.Consequently,the thickness of mammillary and effective layers,and eggshell mechanical properties declined(P < 0.05).LPS treatment downregulated duodenal CALB1 m RNA expression,and reduced calcium and phosphorus contents in eggshell,serum and uterus(P <0.05).LPS injection resulted in infiltration of lymphocytes and edema or dissolution of tubular glands,accompanied with decreased villus height,mucosal fold height and mucosal fold area(P < 0.05).The increased expression of IL-1??TNF-??occludin?ZO-1?ovotransferrin and ovalbumin,and the declined expression of CALB1 and osteopontin were observed in uterus of LPS-treated group(P < 0.05).These findings indicated that LPS-induced inflammation could destroy the uterine morphology,obstruct the supply of calcium and phosphorus,and then interfere with calcium transport and matrix protein synthesis in uterus,possibly resulting in abnormal ultrastructure and declined mechanical properties of eggshell.The aim of experiment 3 was to explore whether dietary essential oil supplementation(50,100 and200 mg/kg)could enhance uterine health and biomineralization-associated functions,thereby conducing to the improved ultrastructure and mechanical properties of eggshell in late-phase laying hens.Results showed that essential oil addition linearly and quadratically elevated thickness,breaking strength,weight and the ratio of eggshell,and quadratically increased eggshell stiffness(P < 0.05).There was a linear and quadratic increase(P < 0.05)in effective thickness and eggshell thickness.The addition increased the incidence of early fusion and cuffing in mammillary layer and decreased the incidence of type B mammillae and total score for mammillary variants(P < 0.05).These beneficial effects were maximized with 100 mg/kg essential oil addition and then further analysis was conducted on control and 100 mg/kg essential oil group.Dietary addition resulted in the elevated duodenal CALB1 expression,serum calcium levels and uterine villus height(P < 0.05),with a tendency to reduce the ratio of edema or dissolution of tubular glands(P = 0.088).Essential oil addition depressed the expression of IL-1? and IL-6 in uterus and reduced inflammatory cell infiltration(P < 0.05).The expression of ion transport-associated genes CALB1,ATP2B1,ATP2B2 and SLC26A9 was increased,whereas the expression of matrix proteins ovalbumin and ovotransferrin was decreased in uterus in response to dietary essential oil supplementation(P < 0.05).These findings indicated that dietary essential oil supplementation could improve uterine morphology and inflammatory immune status,regulate the expression of ion transport and matrix protein genes,resulting in decreased percentage of abnormal structures in mammillary layer and increased thickness of effective layer and eggshell,which may contribute to the elevated breaking strength and stiffness of eggshell.In summary,the altered gene expression of matrix proteins along with the compromised immune function in uterus of late-phase laying hens may conduce to the compromised mammillary layer structure and the elevated thickness of mammillary layer;dietary supplementation with essential oil could improve uterine morphology and inflammatory immune status,and regulate uterine biomineralization-associated functions,which may contribute to the enhanced ultrastructure and mechanical properties of eggshell in the late laying period.