| Cage layer osteoporosis(CLO)is a common bone metabolic disease in high-yield laying hens,resulting in great susceptibility to fractures and paralysis,producing a large number of sand shell eggs and soft shell eggs,and causing a broken egg rate up to 0.5%-1%.A preliminary survey of large-scale laying hen farms in seven provinces of China found that the incidence of CLO was between 0.1% and 0.3%.As cage layer is still the main breeding mode in China,CLO has seriously affected the economic benefits of laying hen industry.However,so far,there is no effective precaution for CLO.Icariin(ICA),the main bioactive component of Chinese herb Epimedium,has shown good therapeutic effects on osteoporosis in humans,ovariectomized mouse/rats,glucocorticoid-induced rats and osteoprotegerin knockout rats.However,it is unclear whether ICA also has a preventive effect on CLO.In this study,older caged laying hens(54-66 week of age)fed a basal diet and young caged laying hens(32-40 week of age)fed a low-calcium diet were selected and supplemented with different levels of ICA in the diet.The preventive effects of ICA on CLO under normal physiological conditions and pathological conditions of laying hens were investigated,and the relevant mechanisms of ICA on preventing CLO were revealed by using untargeted metabolomics analysis.This study consists of two animal experiments.The main research contents and results are as follows:Animal experiment 1,Effects and mechanisms of ICA on bone health in older caged laying hens(54-66 week of age):In this experiment,a total of 216,54-wk-old Lohmann pink-shell laying hens were randomly allocated to 3 groups.The control group was fed a basal diet.The treatment group 1 and treatment guoup 2 were fed a basal diet supplemented with 0.05% or 0.20% ICA,respectively.Effects of ICA on bone health of older caged laying hens were investigated,and the relevant mechanisms were explored by using untargeted metabolomics.The main results ale as follows:(1)Dietary supplementation with 0.20% ICA significantly increased average egg weight of older caged laying hens.At 66 week of age,ICA significantly increased albumen height and haugh unit.(2)Dietary supplementation with 0.20% ICA significantly increased the bone mineral density of femur and tibia in older caged laying hens,and improved bone histomorphometry parameters,including increased cortical width,trabecular area ration and trabecular thickness,and reduced trabecular separation.(3)Dietary supplementation with 0.20% ICA reduced the activity of alkaline phosphatase,tartrate-resistant acid phosphatase,and osteocalcin content in serum of older caged laying hens.(4)Dietary supplementation with 0.05% ICA or 0.20% ICA both significantly increased the expression of osteogenesis-related genes RUNX2 and OPG,and decreased the expression of osteoclast-related genes RANKL in the femur tissue of older caged laying hens.(5)Serum untargeted metabolomics analysis screened out 170 differential metabolites between the control group and the treatment 2 group.Furthermore,Spearman correlation analysis between the differential metabolites and the bone mineral density of femur and tibia in older caged laying hens found that 8 differential metabolites were highly both correlated with the bone mineral density of femur and tibia,including uridine,taurine,palmitic acid,adrenic acid,fexofenadine,lyso PC(18:1),lyso PE(20:3/0:0)and 3-acetyl-11-keto-beta-boswellic acid.Then,the metabolic pathway analysis of the above 8 differential metabolites by Metabo Analyst 4.0 revealed that ICA mainly perturbed taurine and hypotaurine metabolism,glycerophospholipid metabolism and pyrimidine metabolism.Animal experiment 2,Effects and mechanisms of ICA on preventing low calcium diet-induced osteoporosis in young cage laying hens(32-40 week of age):In this experiment,a total of 144,32-wk-old Lohmann pink-shell laying hens were randomly allocated to 4 groups.The control group was fed a basal diet(3.5% Ca).The low calcium diet group was fed a low calcium diet(2.0% Ca).The treatment group 1 and treatment guoup 2 were fed a low calcium diet supplemented with 0.05% or 0.20% ICA,respectively.The preventive effects of ICA on low calcium diet-induced CLO in young caged laying hens were investigated and the relevant mechanisms were explored by using untargeted metabolomics.The main results are as follows:(1)Compared to the control group,low calcium diet caused a significant decrease in production performance and egg quality of young caged laying hens.Low calcium diet supplemented with ICA improved production performance.(2)Compared with low calcium diet group,low calcium diet supplemented with 0.20% ICA significantly increased femur bone mineral density,cortical width,trabecular area ration and trabecular thickness,and reduced trabecular separation in young caged laying hens.(3)Compared with low calcium diet group,low calcium diet supplemented with 0.20% ICA significantly reduced the activity of alkaline phosphatase,tartrate-resistant acid phosphatase,and osteocalcin content in serum of young caged laying hens.(4)Compared with low calcium diet group,low calcium diet supplemented with 0.05% ICA or 0.20% ICA both significantly increased the expression of osteogenesis-related genes RUNX2 and OPG,and decreased the expression of osteoclast-related genes RANKL in the femur tissue of young caged laying hens.(5)Screened by serum untargeted metabolomics analysis,8 metabolite levels were significantly changed with ICA treatment,including 7-dehydrocholesterol,7-oxocholesterol,desmosterol,PC(18:1(9Z)/18:1(9Z)),PS(18:0/18:1(9Z)),N,N-dimethylaniline,2-hydroxy-butanoic acid and N2,N2-dimethylguanosine.Further,metabolic pathway analysis of the above 8 metabolites through Metabo Analyst 4.0 found that ICA mainly disturbed the steroid biosynthesis and glycerophospholipid metabolism.In summary,the main conclusion of this study is:(1)Dietary icariin supplementation produced osteoprotective effects on older caged laying hens and effectively relieved bone loss;the bone mineral density of older caged laying hens was positively correlated with serum uridine,taurine,palmitic acid,adrenic acid,fexofenadine,lyso PC(18:1)and lyso PE(20:3/0:0)concentration,and was negatively correlated with serum 3-acetyl-11-keto-beta-boswellic acid concentration;icariin mainly produced osteoprotective effects by perturbing taurine and hypotaurine metabolism,glycerophospholipid metabolism and pyrimidine metabolism.(2)Icariin relieved low calcium diet-induced osteoporosis in young caged laying hens;low calcium diet supplemented with icariin caused serum 7-dehydrocholesterol,7-oxocholesterol,desmosterol,PC(18:1(9Z)/18:1(9Z)),PS(18:0/18:1(9Z)),N,N-dimethylaniline and 2-hydroxy-butanoic acid concentration significantly increased,and serum N2,N2-dimethylguanosine concentration significantly decreased;icariin relieved osteoporosis in young caged laying hens mainly by perturbing steroid biosynthesis and glycerophospholipid metabolism.The osteoprotective effects of icariin,and the discovery of endogenous metabolites correlated with bone mineral density will provide potential nutritional regulation strategies for the prevention of cage layer osteoporosis. |
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