The Mechanisms Of Age And Letrozole On Bone Metabolism In Laying Hens

Bone mainly consists of about 55 wt.%mineral,30 wt.%collagen,and 15 wt.%water.Mineral(bioapatite)is the primary component in bone,which deeply affects bone functions and pathology.Bioapatite(BAp)is actually a type of carbonated hydroxylapatite(carbonate mostly substituting for phosphate).Coupled with the substitution of CO32-for PO43-in BAp,Na,Mg,or other minor/trace elements can substitute for Ca cations,which causes Ca depletion in bone in addition to the resorption of whole mineral crystals.In mineralogy,increasing carbonate substitution usually leads to a lower degree of crystallinity in apatite.Bone metabolism is regulated by calcium-regulated hormones and sex hormones.In order to explore the changes of bone metabolism in laying hens and the influences of estrogen suppression,laying cycle model in birds was established by four special periods and anti-estrogen drugs treatment to study its mechanism.Understanding the details of bone mechanism and Ca loss in hens can provide evidences for further studies about the Ca deficiency and bone degradation during egg-laying period.Experiment ? Changes of Bone Mineralogy of Layers in Different StagesIn this study,eight Gushi layers were selected randomly in the four periods:before sexual maturity(11 w),sexual maturity(18 w),peak time of egg laying(49 w)and end of laying period(77 w),measuring the changes of bone mineralogy.After being weighted and harvested 5 mL blood in wing vein,those Gushi layers were killed according to the experimental instruction.Then femur and tibia bones were collected to determine their bone densities,bone intensities and the mineralogical changes in the cortical bones by Raman spectroscopy.The results showed that the bone density of Gushi layers increased in an age-dependent manner from 11 w to 77 w,and the bone mineral density of the latter three groups was significantly higher(P<0.05)than that of the 11 w.Instead,the bone density of bone increased slowly at 49 w and that of tibia was decreased.The bone strength of the humerus,femur and tibia kept an increasing trend with age during laying period,but the results of the 49 w showed a significant decrease(P<0.05)compared with that of the 18 w.Raman analysis showed that the crystallinity of the humerus,femur and tibia of the laying hens decreased significantly,and the carbonate contents did highly increase during laying period(P<0.05).It was showed that laying did significantly affect the bone mineral density and strength of layers.Experiment ? Preliminary Studys on the Changes of Bone Metabolism with Age in LayersLaying hens(Gushi layer)at stages of age(8 hens each stage)were studied:before sexual maturity(11 w),sexual maturity(18 w),peak time of egg laying(49 w)and end of laying period(77 w).The levels of blood biochemical markers of Ca,P,StrACP,ALP and BGP,Ca-regulated hormone of PTH and CT,and sex hormone of E2 were determined in order to explore the related regulation on bone metabolism in laying hens.The results showed that the plasma Ca level did significantly increase at 18 w and 49 w,but the level of 77 w was significantly lower than that of 49 w when compared with the 11 w(P<0.05).However,the change of plasma P level had no significant difference during the experiment.After 18 weeks,there was a significant decrease in ALP and BGP activities compared with 11 w layers(P<0.05).The level of PTH concentration at 49 w was significantly higher than that of birds at 11 w and 77 w.The level of CT at 49 w was significantly higher than that of the other three stages(P<0.05).The E2 of sex hormones also changed significantly.Compared with the 11 w,the concentration of E2 at 18 w significantly increased,reached the highest peak when at the age of 49 w,and significantly lowered at 77 w(P<0.05).These results showed that there were a series of changes on bone metabolism during the laying cycle of laying hens,which was closely related to the regulations of Ca and P metabolism,Ca-regulated hormones and sex hormones.Experiment ? Regulation of Letrozole on Bone Metabolism in Young LayersIn this study,the effects of letrozole on changes of bone metabolism in young layers were studied.After one week of being perfed,200 laying hens at the age of 13 weeks old were randomly divided into two groups(the control and letrozole group)before experiment.The control and experiment group were fed with 4 mL/d normal saline and letrozole(LZ)0.5 mg/d,respectively.The experimental period lasted for 9 weeks.Blood samples were collected from two groups(N=8)at the 3-,6-and 9-week-hen respectively,in order to determine the changes of the bone mineral metabolism markers of StrACP,ALP and BGP,Ca-regulated hormone of PTH and CT,and sex hormone of estradiol and testosterone.Bone samples were measured for their bone densities and strengthes.The results showed that both the bone mineral densities and strengthes of the humerus,femur and tibia significantly increased in the letrozole group(P<0.05).The levels of serum calcium did highly decrease in the letrozole group at 3 w and 6 w(P<0.05),but serum P concentrations did not change significantly.The activity in StrACP of bone markers was significantly higher than that in the control group,but there was no significant difference between the letrozole and control group.The ALP activity significantly decreased at 9 w and the BGP levels highly decreased at 6 w and 9 w(P<0.05).Compared with the control group,the level of PTH,a bone-metablism-regulated hormone,was significantly lower than that in the experiment group,and the CT level did not obviously change.The E2 levels of 6 w and 9 w significantly increased compared with the 3 w with the age,and the E2 level of treated group was significantly lower than the control group.However,T1 hormone in treated group demonstrated the opposite trendency compared with the control group(P<0.05).The results showed that letrozole significantly reduced plasma calcium,StrACP and estrogen levels,elevated the levels of ALP,BGP and androgen,but did no significantly affect the CT level.Meanwhile,it also increased bone density and strength.Experiment IV Regulation of letrozole on Bone Metabolism in Young LayersIn the process of bone metabolism,effects on calcium-processing genes(CaBP-D28K,TRPV6,NCX1 and PMCA lb),calcium-regulating hormone receptor genes(PTH1R and CALCR)and sex hormone-related receptors genes(ESR2 and TR)in the duodenum and kidney of young laying hen with letrozole treatment were studied in this study.At the same time,the expressions of CaBP-D28K protein in the duodenum and kidney were studied in the protein level.The results showed that letrozole significantly down-regulated the transcriptional level of duodenal CaBP-D28K and up-regulated the transcription level of PTH1R,but there had no effect on other genotypes of duodenum in the 3-week-LZ-treated hens.Meanwhile,letrozole had significantly up-regulated the mRNA levels of renal calcium-regulating hormone and sex hormone-related receptor genes.At 6 w,letrozole had significantly down-regulated the levels of CaBP-D28K,ESR2 and CALCR mRNA up-regulated NCX1,PMC A lb and TR mRNA in the duodenum,and significantly up-regulated the levels of CaBP-D28K,NCX1,PMC A lb and CALCR mRNA in the kidney.At 9 w,letrozole had significantly down-regulated the transcriptional levels of the duodenal CaBP-D28K,PMCA lb,ESR2 and CALCR and up-regulated TR transcription.However,letrozole had significantly up-regulated the renal transcription of TRPV6 and NCX1,and down-regulated the level of PMCA 1b mRNA,but had no significant effect on other gene transcription.At the level of protein expression,letrozole significantly reduced the expressions of CaBP-D28K protein in the duodenum,but had no significant change in the kidney.The results showed that letrozole significantly reduced the expression of CaBP-D28K at the late stage from the gene and protein levels and decreased the absorption of calcium in the duodenum.While bone metabolism was affected by the expression of PTH1R and CALCR,and it was also indirectly regulated by ESR2 and TR.