Analysis Of The Mechanism Of Laying Hens Adapting To Low-phosphorus Diet Based On FGF23 Signaling Pathway

The resource and environmental problems caused by the excessive use of non-renewable inorganic phosphate rock are an important topic of concern in the livestock industry.A large number of studies have shown that the actual requirement of phosphorus in the diet of laying hens is far lower than the recommended amount in the industry’s conventional nutrition recommendation system（such as NRC1994 and China Chicken Feeding Standard 2004）.However,in actual production,the vast majority of laying hen farmers maintain a cautious and conservative attitude towards low-phosphorus diets,and the level of dietary phosphorus remains high.The core reason is that mechanism of phosphorus diets are not accurately recognized in laying hens.In this study,diets with different levels of non-phytate phosphorus were set for laying hens,with the body’s phosphorus nutrition process as the main line,based on the fibroblast growth factor 23（FGF23）signal pathway,and analyzing internal mechanism for laying hens to adapt to low-phosphorus diets.This work independently developed fibroblast growth factor receptor 1（FGFR1）monoclonal antibody to verify the adaptation mechanism.The relevant results can promote the industry to rethink the dietary phosphorus levels of laying hens.Experiment 1:Effects of different non-phytate phosphorus levels on phosphorus nutritional status of laying hensIt is generally reported in the literature that the non-phytate phosphorus level of0.14-0.42%in the diet has no significant effect on the production performance and egg quality of laying hens,but it has not been explained by a comprehensive analysis of the phosphorus nutritional status of the body.In this experiment,14 Hylan brown layer hens（38weeks old）were randomly divided into two treatment groups（n=7）,and the non-phytate phosphorus levels were 0.14（phosphorus restriction group）and 0.32%（regular phosphorus group）corn-soybean meal type diet,the feeding trial lasts 21 days.During the test,the feed intake and egg weight of the chickens were recorded daily;on the 21st day of the test,the egg quality was measured,and the whole-day excrement of each chicken was collected separately;on the 22nd day of the test（08:00 in the morning）,the slaughter was concentrated,Collect blood,intestine and kidney samples for analysis of phosphorus nutrition status of chickens under different dietary phosphorus supply modes.The results showed that diets with different levels of non-phytate phosphorus had no significant effect on the production performance and egg quality of laying hens（P>0.05）.Production performance indicators include egg production rate,egg weight,daily feed intake,feed-to-egg ratio and Body weight before and after the test,egg quality indicators include eggshell thickness,eggshell index,eggshell strength,egg white height,egg yolk color,Haugh unit and egg specific gravity.There was no significant difference in blood calcium level and alkaline phosphatase activity between the two treatment groups（P>0.05）.Compared with the normal phosphorus group,the blood phosphorus level of laying hens in the phosphorus restricted group was significantly reduced（P<0.001）,and the protein expression of 2a type sodium phosphorus co-transport carrier（NPt2a）in the kidney was significantly reduced（P<0.05）,protein expression of duodenal 2b type sodium phosphorus co-transport carrier（NPt2b）increased significantly（P<0.05）,and phosphorus excretion decreased significantly（P<0.05）.Conclusion of this experiment:After reducing the level of non-phytate phosphorus in the diet,laying hens can improve the intestinal phosphorus absorption efficiency to maintain the phosphorus status of the body.Although the blood phosphorus level has been lowered,it has no negative impact on production performance and egg quality.Experiment 2:Effects of different non-phytate phosphorus levels on FGF23 signal of laying hensFGF23,a newly discovered phosphorus-regulating factor in 2000,is considered to be the core hormone for the regulation of phosphorus homeostasis.However,FGF23 has little research on the regulation of phosphorus homeostasis in laying hens.At the end of the experiment,blood,skull,liver,kidney and intestine samples were collected for the determination of the expression of FGF23 and its receptors（FGFR1,FGFR2,FGFR3,FGFR4 and Klotho）,which aim to analyze the changes in different dietary phosphorus supply patterns,by the FGF23 signalling network of laying hens and their contribution to adapting to low-phosphorus diets for laying hens.The results showed that the blood FGF23levels of the chickens in the phosphorus restriction group were significantly lower than those in the regular phosphorus group,and the m RNA expression levels of the skull FGF23 and FGFR1 in the phosphorus-restricted group,the m RNA expression levels of the kidney FGFR1 and FGFR4,and the m RNA expression of the jejunum FGFR4 The amount was significantly reduced（P<0.05）.There was no significant difference in the levels of parathyroid hormone（PTH）and 1,25-Dihydroxyvitamin D₃（1,25（OH）₂D₃）in the blood between the two treatment groups（P>0.05）.Conclusion of this experiment:Reducing the dietary non-phytate phosphorus level,laying hens downregulate the expression of FGFR1and FGF23 in the skull,reduce the blood FGF23 concentration,further inhibit FGF23signaling in the kidneys and intestines,reduce the body’s phosphorus loss,and then adapt to low phosphorus diet.Experiment 3:Effect of FGFR1 antibody on phosphorus nutritional status of laying hensThe results of Experiment 1 and Experiment 2 suggest that laying hens use FGFR1 to adjust FGF23 signal transduction to adapt to changes in dietary phosphorus levels.In this experiment,FGFR1 activating antibody was independently constructed to verify the role of FGF23 signaling pathway in maintaining phosphorus homeostasis of laying hens.21 Hailan brown layer hens（30 weeks old）were randomly divided into 3 groups（n=7）,respectively:（1）control group（injected vaccine only contains adjuvant）;（2）Cr Z-1 vaccine group（injected vaccine contains chicken FGFR1 protein molecule surface antigen fragment Cr Z-1:LPEDPRWE,which can induce the body to produce FGFR1 monoclonal activation antibody）;（2）Cr Z-2 vaccine group（injected vaccine contains chicken FGFR1 protein molecule Surface antigen fragment Cr Z-2:LDKDKPNR,which can induce the body to produce FGFR1 monoclonal activation antibody）.The vaccination time is 1,14 and 28 days of the test.According to the previous study,the antibody titer reached the peak on the 35th day of the test.Therefore,on the 35th day of the experiment,the whole day excreta of chickens were collected separately,serum samples were collected,and the quality of eggshells was measured to analyze the phosphorus nutritional status.The results showed that:between each treatment group,production performance（including egg production rate,egg weight,daily feed intake and feed-to-egg ratio）,eggshell quality（including eggshell thickness,eggshell strength and eggshell index）and phosphorus excretion status There was no significant difference（P>0.05）.However,compared with the control vaccine group,the blood phosphorus level of the chickens in the Cr Z-1 vaccine group had a tendency to decrease（P=0.075）,and the blood phosphorus level of the chickens in the Cr Z-2 vaccine group decreased significantly（P<0.05）.Conclusion of this experiment:After activating FGF23 signaling pathway with FGFR1 antibody,the blood phosphorus level of laying hens was significantly reduced.This result verified the role of FGF23 signaling in regulating the blood phosphorus level of laying hens and maintaining the body’s phosphorus homeostasis.This study shows that short-term supply of 0.14%non-phytate phosphorus level diets has no adverse effect on the performance and egg quality of laying hens.Chickens can adapt to low-phosphorus diets by reducing blood phosphorus levels,inhibiting the FGF23"phosphorus-removing"signal network,strengthening the intestinal phosphorus absorption capacity,and maintaining the phosphorus retention efficiency of the body.This study analyzes the adaptability of laying hens to low-phosphorus diets based on the FGF23 signal,and answers the physiological basis of laying hens to adapt to low-phosphorus diets in the literature,which can provide new considerations for the preparation of phosphorus-element resources and environmentally-friendly diets.