Effects Of Vitamin D Level,Source And Feeding Stage On Production Performance And Bone Quality Of Laying Hens And Possible Mechanisms

Vitamin D（VD）is an important vitamin to maintain the bone health and eggshell quality of laying hens.In recent years,the recommended VD requirement of laying hens has changed greatly,and the VD requirement is worth further study.In addition,studies on VD requirements of laying hens mainly focused on the laying stage,so it is worth further exploring whether VD levels can improve the performance and bone quality of laying hens by increasing the levels of VD in rearing stage and whole stage.Vitamin D₃（VD₃）and 25-hydroxyvitamin D₃（25-OHD）are common sources of VD in poultry diets.25-OHD,as a metabolite of VD₃,it is worth further study how to use it in combination with VD₃.Based on this,the purpose of this study:through five experiments,to study the effect of dietary VD deficiency on the laying performance of laying hens in the peak period,and to evaluate the demand for VD₃ and the relative biological value of 25-OHD;To study whether it can improve the bone growth of laying hens at the rearing stage by increasing the level of dietary VD at the rearing stage and the whole period,and different combinations of VD₃ and 25-OHD,so as to improve the laying performance at the laying stage,and explore its possible mechanism.Experiment 1.Requirements of VD₃ and relative bioavailability of 25-Hydroxy D₃ in laying hens during peak periodThe purpose of this experiment was to study the requirement of VD₃ and the relative biological value of 25-OHD in laying hens.The experiment included two stages:VD depletion stage and VD supplement stage.VD depletion stage:A total of936 Lohman laying hens（36 weeks of age）were fed a basal diet without VD for 5weeks.VD supplement stage:A total of 528 VD depletion hens were randomly allotted into a 2×5+1 factorial design with two VD sources（VD₃ and 25-OHD）and five levels of dietary VD（75,150,225,300 and 1600 IU/kg）and a negative control group（without VD）.The experiment lasted for 8wk.The results showed as follows:In the VD depletion stage,with the extension of VD depletion time,the laying rate was decreased rapidly from the 3^th week,and the broken eggs rate and soft-shell eggs rate were increased rapidly;Egg quality（eggshell strength,eggshell thickness,eggshell relative weight,haugh unit,albumen height and yolk color）was decreased rapidly;The contents of serum calcium（Ca）,phosphorus（Pi）and 25-OHD were decreased significantly,and the activities of serum alkaline phosphatase（ALP）and parathyroid hormone（PTH）content were increased significantly（P<0.05）.In the VD supplement stage,compared with the control group（VD 0 IU/kg）,the addition of VD3 and 25-OHD in the diet significantly increased the hen-day laying rate,hen-housed laying rate,qualified egg rate,egg weight and feed intake,and significantly reduced FCR,significantly improved eggshell strength（4wk）,eggshell thickness（4 and 8wk）,eggshell relative weight（4 and 8wk）,albumen height（4wk）,haugh unit（4wk）,yolk color（4wk）and yolk relative weight（4wk）,significantly reduced serum ALP activity and Ca content（in addition to adding VD₃）,significantly increased tibial strength and reduced tibial Pi content（P<0.05）.The results of two factor analysis:compared with VD₃ group,the hen-day laying rate（5-8wk）and hen-housed laying rate（5-8wk）in 25-OHD group were increased significantly,and FCR（5-8wk）was decreased significantly,the increase of VD level significantly increased the qualified egg rate,egg weight and feed intake at 5-8wk,and significantly reduced FCR at 5-8wk（P<0.05）.The eggshell strength,eggshell thickness and eggshell relative weight in 25-OHD group were increased significantly at the 4^th wk compared with VD₃ group;Compared with VD 75 IU/kg group,the increase of VD level significantly increased the eggshell strength,eggshell thickness,eggshell relative weight,albumen height and yolk relative weight at the 4^th and 8^th wk（P<0.05）.Compared with VD3 group,the activity of serum ALP and the content of PTH in 25-OHD group were decreased significantly;Compared with VD 75 IU/kg group,the activity of serum ALP in VD 225,300 and 1600 IU/kg group was decreased significantly（P<0.05）.Dietary VD level and source had no effect on tibial strength,tibial ash content,tibial Ca content and tibial Pi content（P>0.05）;Compared with VD 75 IU/kg group,the content of tibial Pi in VD 150,300 and 1600IU/kg group was decreased significantly（P<0.05）.The biological value of 25-OHD relative to VD3 were 139.0～173.3 when the laying performance（5-8wk;hen-day laying rate,hen-housed laying rate,egg weight,FCR and qualified egg rate）was used as the evaluation index;when the eggshell quality（4wk;eggshell strength,eggshell thickness and eggshell relative weight）was used as the evaluation index,the relative biological value was 167.4～221.2;when the eggshell quality（8wk;eggshell strength,eggshell thickness and eggshell relative weight）was used as the evaluation index,the biological value of 25-OHD relative to VD₃ were 108～228.1.When the hen-day laying rate（5-8 wk）was taken as the measurement index,the requirements of VD₃and25-OHD were 254.1 and 146.7 IU/kg respectively;When the hen-housed laying rate was taken as the measurement index,the requirements of VD₃ and 25-OHD were312.7 and 189.2 IU/kg.To sum up,the hen-day laying rate,hen-housed laying rate and egg weight are taken as the evaluation index,the biological value of 25-OHD relative to VD3 is 170,it is close to the biological value 173 determined by the eggshell strength（4wk）and eggshell thickness（4wk）.When the hen-day laying rate（5-8 wk）and hen-housed laying rate（5-8wk）were taken as the measurement index,the requirement of laying hens for VD3 was 254.1 and 312.7 IU/kg,and the requirement of 25-OHD was 146.7 and 189.2 IU/kgExperiment 2.Effect of dietary vitamin D levels and sources in the laying breeder hens diet during 12-20wk on growth performance,bone quality and laying performance in the laying stageThe purpose of this experiment was to study the effects of increasing the levels of VD₃ and 25-OHD in the diet of laying hens aged 12～20 weeks on growth performance,bone quality and laying performance.A total of 1760 12-week-old Lohman pullets were randomly allotted in a 2×2 factorial design with 2 levels of dietary VD₃（300 and 2800 IU/kg）and 2 levels of dietary 25‐OHD（0 and 56μg/kg）.At 19 wk,10 cocks were added to each replicate.Birds were fed the trial diets for 8weeks from 12 to 20 weeks of age.After 20 wk,all hens were fed the same diet,and the laying performance and egg quality were investigated at 30,40,50 and 60 wk.The results showed that the level of VD₃ and the addition of 25-OHD in 12-20 wk diet had no significant effect on the growth performance of laying hens（P>0.05）;Increasing VD₃（2500 vs.300 IU/kg）and adding 25-OHD 2760 IU/kg improved the body weight uniformity of hens at 20 wk（BWU,P<0.05）.Increasing the level of dietary VD₃ significantly increased the content of serum Ca at 20 wk（P<0.05）.The addition of 25-OHD 2760 IU/kg significantly decreased the activity of serum ALP and the number of tibial osteoclasts and significantly increased the contents of Ca,Pi and ash in tibia at 20 wk（P<0.05）.Micro-CT results showed that the addition of25-OHD 2760 IU/kg significantly increased the medullary bone volume（BV）,medullary bone mineral content（BMC）and total bone BMC（P<0.05）,the addition of 25-OHD had no effect on the quality of cortical bone and trabecular bone in tibia（P>0.05）.The level of dietary VD₃ and the addition of 25-OHD had no significant effect on the laying performance and eggshell quality（P>0.05）;Adding 25-OHD2760 IU/kg in 12-20 wk diet increased albumen height（50wk）and haugh unit（50wk）（P<0.05）;There was a significant interaction effect between dietary VD3 level and25-OHD on percentage of setting eggs at 30 wk（P<0.05）.The addition of 25-OHD to VD₃300 IU/kg diet significantly improved the percentage of setting eggs,but the addition of 25-OHD to VD₃ 2500 IU/kg diet had no effect on percentage of setting eggs.Adding 25-OHD 2760 IU/kg significantly increased the tibial bone mass at 20wk and the ash content of tibia at 60 wk.The above results showed that increasing the dietary VD3 level or adding 25-OHD had no significant effect on the growth performance of laying breeder hens,and could not improve the laying performance and eggshell quality at 30,40,50,and 60 wk.However,the addition of 25-OHD 2760IU/kg in 12-20 wk diet increased the tibial bone mass at 20wk and tibial ash content at60 wk.Experiment 3.Effect of dietary vitamin D levels and sources in the laying hens diet during 1-20wk on growth performance,bone quality and laying performance in the laying stageThe study was conducted to evaluate the effect of 25‐hydroxyvitamin D₃（25-OHD）with different levels of VD₃ in the rearing period（1-20 weeks）on growth performance,bone quality and laying performance,and further clarify that improving the dietary VD3 level and adding 25-OHD can improve the production performance and bone quality in the laying stage.A total of four hundred 1‐day‐old Lohman pullets were randomly allotted into a 2×2 factorial design with 2 levels of dietary VD₃（300 and 2800 IU/kg）and 2 levels of dietary 25‐OHD（0 and 2240 IU/kg）during1-20wk.Each treatment had five replicates of 20 hens each.All treatments were fed the same diet for 21-72 wk to further investigate the performance of laying hens.The results showed that:1)the level of dietary VD3 and the addition of 25-OHD had no significant effect on the growth performance of 1-19 wk laying hens（P>0.05）.Compared with VD₃ 300 IU/kg group,the BWU at 8wk in VD₃2800 IU/kg group was improved significantly（P=0.079）.Increasing the level of dietary VD₃（2800 vs 300IU/kg）significantly increased the shank length（SL）at 2,3,4 and 5 wk（P<0.05）.The addition of 25-OHD 2240 IU/kg to the diet during 1-20 wk reduced the mortality of 21-72 wk and 1-72 wk（P=0.030,0.058）,and increased the SL of layers at 7 wk（P<0.05）.There was a significant interaction between dietary VD3 level and 25-OHD on BWU at 8wk（P<0.05）.The addition of 25-OHD in VD₃ 300 IU/kg diet tended to improve the BWU at 8 wk,but the addition of 25-OHD in VD₃ 2800 IU/kg diet had no effect on BWU.Dietary VD₃ level and 25-OHD had no significant effect on 19 wk SL（P>0.05）.By fitting the curve of SL and week age of laying hens in each group,it was found that the SL of laying hens reached the maximum at about 18 wk,which was about 102 mm.2)Compared with 300 IU/kg VD₃ group,the 2800 IU/kg VD₃group showed higher keel length at 10 weeks（P<0.05）.The addition of 25‐OHD significantly increased the keel calcified rate at 20 wk（P<0.05）.3)Increasing dietary VD₃ level（2800 vs.300 IU/kg）increased tibia ash and phosphorus content at 10 wk（P<0.05）.Dietary supplementation with 25-OHD increased tibia ash content at 10weeks,tibia calcium content and tibia strength at 20 weeks（P<0.05）.Increasing dietary VD₃ level（2800 vs.300 IU/kg）increased tibial bone mass（BMD and BMC）,medullary bone mass（BMD and BMC）and total bone mass（BMD,BV and BMC）at20 weeks.The addition of 25-OHD significantly increased the BMD and BMC（cortical bone,medullary bone,trabecular bone and total bone）and trabecular number（Tb.N）,and decreased the percent of closed porosity（CPP）of cortical bone（P<0.05）.4)Increasing dietary VD₃ level（2800 vs.300 IU/kg）increased the serum calcium concentration at 20 weeks and Bone-specific alkaline phosphatase（BALP）concentration at 10 and 20 weeks,and decreased the number of osteoclasts in tibia（P<0.05）.The addition of 25-OHD significantly increased serum Ca concentration（10wk,20wk）and 25-OHD concentration（10wk,20wk）（P<0.05）,and the expression of dentin matrix protein（DMP1）,sclerostin（SOAT）,phosphodiesterase 1（ENPP1）and osteoprotegerin（OPG）m RNA in tibia（P<0.05）,and decreased the content of tartrate-resistant acid phosphatase（TRAP）and C-terminal peptide of type I collagen（CTX）in serum,the number of osteoclasts in tibia and the expression of V-ATPase m RNA in tibia at 20 wk（P<0.05）.5)The dietary VD₃ level and 25-OHD had no effect on the laying performance（P>0.05）.Compared with 300 IU/kg VD₃group,Dietary 2800 IU/kg VD₃ decreased the egg shape index at 20 weeks and the relative weight of egg yolk at 60 weeks,and increased the albumen height at 40wk（P<0.05）.6)Dietary VD₃ levels（2800 vs.300 IU/kg）increased tibia strength and tibia ash content at 72 weeks;Dietary 25-OHD supplementation increased tibia ash content at 72 weeks（P<0.05）.The above results show that the dietary VD₃ level and adding25-OHD had no effect on the growth performance in the rearing period,laying performance and eggshell quality in the laying period,but could improve the BWU at8wk.The addition of 25-OHD can promote the calcification of tibia and keel,inhibit the bone absorption of tibia,and improve the quality of tibia in the early and late stages of egg laying.Experiment 4.Effect of dietary VD₃ replacement by 25-OHD in the laying hens diet during 1-20wk on growth performance,bone quality and laying performance in the laying stageThe purpose of this experiment was to investigate the effect of dietary VD3replacement by 25-OHD in the laying hens diet during 1-20wk on growth performance,bone quality and laying performance in the laying stage and possible mechanisms.A total of 900 1-day-old Lohman pullets were randomly allotted into three dietary treatments（3 treatments×15 repetitions×20 birds per cage）:2800 IU/kg VD₃;69μg/kg 25-OHD;125μg/kg 25-OHD.After 20 wk,five replicates from each treatment were selected to feed the same commercial feed until 72 wk.The results show that:1)The 5000 IU/kg 25-OHD group had the lowest average daily feed intake（ADFI;1-8,1-19 wk）,body weight（8 wk）,body weight gain（1-8 wk）and shank length（4,7 wk）among all treatments（P<0.05）,but there was no significant difference between VD₃ 2800 IU kg group and 25-OHD 2760 IU/kg group（P>0.05）,the curve fitting of week age and SL showed that the SL of laying hens reached the maximum at 17 wk,about 103 mm.2)Compared with VD₃ 2800 IU/kg group,the content of serum PTH in 25-OHD 2760 and 5000 IU/kg group was decreased significantly at 20wk（P<0.05）,and the content of serum Ca and 25-OHD in25-OHD 5000 IU/kg group were increased significantly at 10wk（P<0.05）;There was no significant difference in serum calcium and phosphorus content（10,20wk）between VD₃ 2800 IU/kg group and 25-OHD 2760 IU/kg group（P<0.05）.3)Compared with 25-OHD 5000 IU/kg group,the keel length of 25-OHD 2760 IU/kg group was increased significantly at 10 wk（P<0.05）.Micro-CT results showed the25-OHD 5000 IU/kg group had the highest the total BMC of tibia among all treatments at 20 wk（P<0.05）.4)Compared with VD₃ 2800 IU/kg group,the expression of SOAT,ENPP1 and OPG m RNA in tibia in 25-OHD 5000 IU/kg group were increased significantly（P<0.05）,and Procollagen type N-terminalpropeptide（P1NP,20wk）,TRAP（10wk）and CTX（10wk）were decreased significantly（P<0.05）.5)There was no significant difference in laying performance during 21-72 wk in each treatment group（P>0.05）.Compared with VD₃ 2800 IU/kg,the eggshell strength in 25-OHD 2760 and 5000 IU/kg groups was increased at 20 wk（P=0.065）;Compared with 25-OHD 2760 IU/kg group,the albumen height（P=0.055）and haugh unit（P<0.05）at 50wk in 25-OHD 5000 IU/kg group were significantly increased.Compared with VD₃ 2800 IU/kg and 25-OHD 2760 IU/kg,adding 25-OHD5000 IU/kg significantly increased the content of ash in tibia at 72 wk（P<0.05）.In conclusion,the addition of 25-OHD 5000 IU/kg in hen diet during the rearing period（1-20wk）reduced the body weight（8 wk）,body weight gain（1-8wk）and feed intake（1-8wk）,inhibited the bone absorption of tibia at 20 wk,promoted the bone mineralization of tibia at 20wk,and increased the total BMC of tibia at 20wk and the ash content of tibia at 72wk.Experiment 5.Effects of dietary high level of VD and its source on production performance and bone quality of laying hens from 1 to 72wkThe study was conducted to investigate the effects of high levels VD diets containing 25-OHD on laying performance and bone quality of laying hens during1-72wk and possible mechanisms.The experiment was followed by experiment 4.20wk laying hens were selected,and the 10 repetitions of each treatment were equally divided into two groups.Two kinds of vitamin D treated diets were fed at 21-72 wk,One is that the level and source of dietary vitamin D are the same as that of 1-20wk（treatment 1,2 and 3:VD₃ 2800 IU/kg,25-OHD 2760 IU/kg and 5000 IU/kg respectively）;The second is to keep the VD3 level at 5000 IU/kg,which is provided by the combination of VD3 and 25-OHD:treatment 4,5 and 6 are VD₃ 5000 IU/kg,25-OHD 2760 IU/kg+VD₃ 2200 IU/kg and 25-OHD 2760 IU/kg+VD₃ 2200 IU/kg respectively.There were 6 treatments in total,and each treatment group had 5replicates.The results showed that:1)there was no significant difference in laying performance among the six treatment groups（P>0.05）.The eggshell strength of treatment 1,3 and 4 groups at 60wk was significantly higher than that of treatment 6groups（P<0.05）;The relative weight of eggshell in treatment 3 group at 60wk was significantly higher than that in treatment 2,4,5 and 6 groups（P<0.05）;The relative weight of egg yolk in treatment group 5 at 40wk was significantly higher than that in treatment groups 2,3 and 4（P<0.05）.2)Compared with treatment 1,the content of serum Ca in treatment 2,4,5 and 6 was increased significantly（P<0.05）,the content of serum 25-OHD in treatment 3,4,5 and 6 layers was increased significantly（P<0.05）,and there was no difference in the content of serum PHT in each group at 72weeks（P>0.05）.3)The tibial strength of treatment 3 and 5 was significantly higher than that of the other four treatments（P<0.05）,and the calcium content of treatment3 and 5 was significantly higher than that of treatment 1（P<0.05）.In addition,the ash content of tibia at 72 wk in treatment group 3 and treatment 6 tended to increase than other groups（P=0.058）.The results of micro-CT showed that the cortical bone mineral density of tibia in treatment 3 and 5 was significantly higher than that in treatment 2,4 and 6（P<0.05）,and the tibial Tb.Th in treatment 3 was significantly higher than that in treatments 1,2,4 and 5（P<0.05）,and tibial Tb.Th in treatment 6was significantly higher than that in treatment 1（P<0.05）.4)The serum P1NP content at 72wk in treatment 2 and 4 was significantly higher than that in treatment 5and 6,and the serum P1NP content in treatment 1 and 3 was significantly higher than that in treatment 5（P<0.05）.The expression of DMP-1 m RNA in treatment 3,5 and6 groups was significantly higher than that in treatment 1,2 and 4 groups（P<0.05）,the expression of SOAT m RNA in treatment 3 was significantly higher than that in treatment 1 and 2,and the expression of SOAT m RNA in treatment 5 was significantly higher than that in treatment 1 group（P<0.05）;There was no significant difference in the m RNA expression of BMP-2,RANKL,OPG,V-ATPase,cathepsin-K and RANKL/OPG in tibia among treatments（P>0.05）.In summary,increasing the level of dietary VD（2800 to 5000 IU/kg）at 1-72 wk could not improve the laying performance and eggshell quality of laying hens.When hens were fed with a total activity of 5000 IU/kg VD diet containing 25-OHD,it promoted the tibia mineralization and increased the tibia strength,tibia calcium content and cortical bone at 72 wk.Conclusion1)When the hen-day laying rate,hen-housed laying rate and egg weight were taken as indicators,the biological value of 25-OHD relative to VD3 was 170,which was close to the biological value 173 determined by eggshell strength and eggshell thickness at the 4th weeks.When the hen-day laying rate（5-8 wk）and hen-housed laying rate（5-8wk）were taken as the measurement index,the requirement of laying hens for VD3 was 254.1 and 312.7 IU/kg,respectively,and the requirement of25-OHD was 146.7 and 189.2 IU/kg2)Dietary VD levels and source during 12 to 20 weeks had no effect on growth performance,laying performance and eggshell quality（30,40,50 and 60 wk）of laying breeder hens.The adding 2760 IU/kg 25-OHD to the diet of 300 IU/kg VD₃during 12 to 20 weeks could increase the bone mass of tibia at 20 wk and ash content of tibia at 60 wk.3)Dietary VD levels and source during 1 to 20 weeks had no effect on growth performance,laying performance and eggshell quality of laying hens.Dietary VD₃level（2800 vs.300IU/kg）and supplemented with 2240 IU/kg 25-OHD can promote tibia mineralization by up-regulating the expression of DPM1,SOAT and ENPP1m RNA,and inhibit bone resorption of tibia by decreasing the number of osteoclasts and down-regulating the expression of RANKL/OPG and V-ATPase m RNA.So as to improve the tibia quality of early and late egg laying.4)The addition of 5000 IU/kg 25-OHD in hen diet during the rearing period（1-20wk）reduced the growth performance of hens from 1-8wk,promoted tibia mineralization by up regulating the expression of SOAT and ENPP1 m RNA,inhibited bone resorption of tibia by down regulating the expression of RANKL/OPG m RNA,and increased the total BMC of tibia at 20 wk and ash content of tibia at 72 wk.5)Feeding hens with high-level VD diet containing 25-OHD for 1-72 weeks can not improve the laying performance and eggshell quality of laying hens,but can promote the tibia mineralization of by up regulating the expression of DMP1 and SOAT m RNA,and improve the tibia strength,tibia calcium content and bone mineral density of tibia at 72 weeks.