Mechanism And Metabonomic Early Warning Evaluation Of Hypophosphatemic Laying Fatigue Syndrome In Laying Hens

Laying fatigue syndrome is a kind of production disease of hens which often occurs in the first laying period and peak laying period.It is also known as osteoporosis in caged laying hens,which is mainly characterized by decreasing laying rate,increasing broken and soft shell eggs,osteoporosis and easy fracture,and death in severe cases.Insufficient phosphorus content in feed is one of the main causes of the disease.According to the biomarkers of laying fatigue syndrome,the disease can be prevented and treated more effectively.Therefore,it is of great significance to establish early warning of hypophosphatemic laying fatigue syndrome.In this experiment,the laying hens model of hypophosphatemic laying fatigue syndrome was established by feeding low-phosphorus diet.On this basis,combined with the method of metabonomics,the occurrence mechanism and early warning indication of laying fatigue syndrome were studied.Sixty 22-week-old laying Roman white shell hens were randomly divided into two groups:control group（group C）and low phosphorus group（group P）.The hens in group P were fed with a mixed diet with available phosphorus content of 0.18%,to establish the model of laying fatigue syndrome.When the hens grew to the age of 25 weeks,29 weeks and 34 weeks,the clinical indexes and laying performance were counted,and the hens sternum were removed and X-rayed.The results showed that compared with group C,the hens in group P could not move flexibly,the legs were bent,it was difficult to stand,even paralyzed,the sternum tissue was obviously bent,the feed intake decreased,the body weight decreased slightly,the laying rate decreased,and the rate of soft shell and broken eggs increased,the thickness and strength of eggshell decreased,and the egg weight decreased,which indicated that the hens had hypophosphatemic laying fatigue syndrome.The serum samples of hens were collected to detect the contents of phosphorus（P）,calcium（Ca）,osteoprotegerin（OPG）,parathyroid hormone（PTH）,estradiol（E₂）,the activities of tartrate resistant acid phosphatase（TRACP）and alkaline phosphatase（ALP）.The results showed that:（1）At the age of 25 weeks,the content of serum P in group P was higher than that of group C（p<0.05）,there was no significant difference between the two groups at 29 weeks,but decreased in group P compared with group C at 34 weeks（p<0.01）.（2）At 25 weeks of age,the serum Ca content of group P was slightly lower than that of group C,but the differ ence was not significant;at 29 weeks of age,the serum Ca content of group P was significantly lower than that of group C（p<0.05）,while at 34 weeks of age,the serum Ca content of group P was higher than that of group C（p<0.05）.（3）At the 25th,29th and 34th week,compared with group C,the activities of serum TRACP and ALP in group P were higher than those of group C（p<0.01）,and the content of serum PTH in group P was higher than that of group C（p<0.01）,while the content of E₂ was lower than that of group C（p<0.01）,the content of serum OPG in group P was also lower than that of group C（p<0.01）.At the same time,the serum samples of the two groups of hens at the 25th,29th and 34th week were tested by LC-MS metabonomics.Eight serum samples were detected in each group,and each sample was tested repeatedly for 10 times to ensure the reliability of the results.The results of non-targeted metabonomics showed that the serum small molecular metabolites in the three periods were enriched in 10 pathways,and 17 metabolites were significantly different by AUC curve analysis.Among them,the cationic metabolites are Fenamole,Parietin,NN,N-Bis（2-hydroxyethyl）dodecanamide,（+/-）-Metanephrine,9-Nitrooleate,Emetine,Auramine,Adrenaline,Biacetyl,ameltolide,Betamipron.The anion metabolites are Dodecyl sulfate,2,4,6-Trichlorophenol,Quingestrone,Dicyclohexyl phthalate,15S-hydroxyeicosatrienoic acid and Trolox.The AUC values of the differential metabolites that were finally screened were all 1,which had extremely high predictive value.In summary,low-phosphorus diet could induce laying fatigue syndrome by affecting calcium and phosphorus content,related enzyme activity and hormone level,10 metabolic pathways and signal transduction pathways of differential metabolites were found in laying hens with laying fatigue syndrome,17 biomarkers were screened out which can be used for early diagnosis and early warning of laying fatigue syndrome in laying hens.This study provides a scientific experimental basis for the early prevention and treatment of hypophosphatemic laying fatigue syndrome.