| Previous studies of our research group showed that glucosamine（GluN）could regulate bone metabolism,but its regulatory mechanism was not yet clear.Therefore,the objective of this study was to determine the effects of GluN on the egg production,egg quality,eggshell quality,bone character,digestion and metabolism,gene expression related to skeleton for exploring the regulation mechanism and providing the theoretical basis to the layers.The study was divided into 3 parts:Experiment1:Effects of GluN on the egg production,egg quality,bone characters and serum biochemical index in laying hens during late of egg production.Single factor experimental design was conducted,and a total of 512 55-week-aged hylline layers were randomly assigned to 4 experimental groups with 8 replicates of 128 layers each group,and were fed with diets supplemented with 0%,0.4%,0.6%and 0.8%s GluN respectively,There were 15 days for pre-test,and trail lasted 60 days.The results showed as follows:（1）egg laying rate and feed intake were improved and abnormal egg rate were reduced with addition of GluN.Compared with 0%group,the total food intake of the 0.4%group increased by 5.87%（P<0.1）;the total food intake of the 0.6%group increased by 8.33%（P<0.1）;the abnormal egg rate reduced by 30.22%（P<0.1）,The positive effect of 0.6%GluN group was more obvious with the extension of test period.（2）GluN increased the eggshell mass,eggshell strength and the calcium mass of eggshell.GluN had the trend of increasing eggshell weight and eggshell strength（P<0.1）at 30th day and 60th day;the calcium mass of eggshell in group 0.4%GluN was significantly higher than that in group 0.6%GluN（p<0.05）at 30th day.the calcium mass of eggshell in group 0.6%GluN was significantly higher than that of group 0.4%GluN（p<0.05）at 60th day.（3）From the test of30th day to 60th day,the tibia mineral density of 0%and 0.4%GluN groups were decreased,but the 0.6%GluN and 0.8%GluN group increased by 44.74%,44.79%,respectively;the mineral density of femur and humerus showed a decreasing trend,but the descending trend of0.6%GluN and 0.8%GluN group was significantly lower than that of 0%and 0.4%GluN group;At 60th day,the young’s modulus,maximum load and maximum bending stress of femur were significantly increased by 64.5%,20.47%and 59.5%comparing with the 0%group,respectively;there were significantly difference with cortical bone thickness,area and percentage of cortical bone area in tibia,cortical bone thickness,area and percentage of cortical bone area of 0.6%GluN group significantly increase by 36.8%,37%,17.12%,respectively;By the result of HE staining from paraffin slices of tibia,Both the0.6%GluN group at 30th day and the 0.8%GluN group at 60th day had a better trabecular continuity than other group.（4）At 30th day,GluN significantly promoted the content of CT,E2 and Ca in serum（P<0.05）,and significantly decreased the IL-6 concentrate（P<0.05）.When 60th day,the secretion of PTH hormones for 0.6%GluN were significantly higher than0.6%GluN group（P<0.05）,and the E2 of 0.6%GluN was more significantly higher than0%GluN（P<0.05）.In addition,GluN had tendency to increase serum Ca level and decrease the content of IL-6,PGE2.Experiment 2:Study of GluN on the calcium absorption in the diet of laying hens.A 2×3factor experiment design was conducted to investigate the effects of GluN on dietary calcium absorption in layers,36 healthy,similar body weight,Hyline-gray layer hens during the late egg production were selected and randomly divided into 6 groups（including 3 0%GluN groups and 3 0.6%GluN group）,3 replications each group,2 hens per replication.Birds were fed with various diets containing 3%calcium,3.2%calcium,3.4%calcium;3%calcium+0.6%GluN,3.2%calcium+0.6%GluN and 3.4%calcium+0.6%GluN.The excreta was collected for 3 days.The apparent metaboblities,the amount of endogenous calcium excretion and ture metaboblities of calcium in the rations were measured.The results showed that:the apparent metabobilities of the controlled and GluN rations are not affected by calcium content,varied from 49.62%to 52.76%and from 62.49%-65.66.Therefore,adding GluN promoted calcium absorption by 11.2%（P<0.05）.The calcium content and GluN level had no significant effects on the amount of endogenous calcium excretion（P>0.05）,the average value was 0.61g per kilogram dry matter intake.True metabolibilites of calcium in rations containing GluN were 81.79%,enhanced by 11.2%compared to controlled group.The expression of Cabp-D28k gene in GluN group was significantly higher than that in control groups（P<0.05）.Therefore,GluN promoted the absorption of calcium by enhancing the expression of Cabp-D28k gene in the duodenum.Experiment3:The effects of GluN on the expression of OPG、RANK、RANKL、CTK、CTR and IL-6 gene in laying hens of the late period of peak egg production.The test design was the same as Experiment 1,at the trial 30th and 60th day,5 laying hens with similar weight were selected from each experimental group,and their left tibia were collected and RNA was extracted.Then,the effects of GluN on the gene expression of bone metabolism related genes OPG,RANK,RANKL,IL-6,protein tissue enzyme K（CTK）and calcitonin receptor（CTR）were investigated by real-time fluorescence quantitative PCR.The result have showed that,（1）GluN up-regulated OPG gene expression,down-regulated RANK and RANKL gene expression,and reduced RANKL/OPG.;At the trial 30th day,the RANKL/OPG ratio decreased with increasing of GluN levels;（2）At the trial 60th day,the expression of IL-6gene of laying hens bone were up-regulated（P<0.05）;（3）In the whole trial period,GluN significantly reduced the expression of CTK gene in 0.6%GluN group（P<0.05）compared with 0%group;（4）In the whole trial period,the expression of CTR gene was increased in0.6%GluN group compared with 0%group.The conclusions were as follows:（1）egg laying rate,feed intake,eggshell weight,eggshell strength were improved and abnormal egg rate were reduced with addition of GluN,The positive effect of GluN was more obvious with the extension of test period;（2）GluN promoted calcium absorption by increasing the expression of Cabp-D28k gene in the intestine of laying hens.（3）GluN increased blood calcium concentration.promoted the secretion of E2,CT,and inhibited the secretion of IL-6,PGE2,PTH,and（4）GluN upregulated OPG and CTR gene expression and inhibited CTK and IL-6 gene expression.（5）GluN increased the continuity of trabecular bone,bone mineral density,bone strength and bone thickness.（6）The regulation mechanism of GluN on bone metabolism:The possible pathway of above-mentioned effects were that GluN promoted calcium absorption by increasing the expression of Cabp-D28k gene in the intestine of laying hens,the concentrate of serum Ca was increased,On the one hand,it increased the calcium content of the eggshell.On the other hand,it increased the concentration of calcium,promoted the secretion of E2 and CT,inhibited the secretion of PTH,then up-regulated the expression of OPG and CTR genes in laying hens,and down-regulated the expression of RANKL,CTK,IL-6 genes and RANKL/OPG.Therefore,activity of osteoclast and degradation of bone matrix was decrease,at the same time the secretion of inflammatory factors IL-6 and PGE2 was restrained,thus reducing the inflammatory effect of the body.Ultimately,the continuity of trabecular bone,bone mineral density,bone strength and bone thickness were increased through the above approaches. |
