| This study was conducted to investigate the modulatory effects of vitamin K on bone quality of broilers by using in vitro culture of osteoblasts isolated from chicken embryos and in vivo feeding experiment of broilers.1.The isolation and identification of osteoblasts from chicken embryosIn a large number of experiments,we used the method of enzyme digestion to improve the method of isolating and culturing osteoblasts from chicken embryos,and performed a series of trails for cell identification.It was confirmed that the isolated cells contained high alkaline phosphatase activity,and could secrete type I collagen enzyme and form calcified nodules.2.Effect of VK2 on the proliferating ability and alkaline phosphatase activity of chicken embryo osteoblastsCells were treated with different levels of VK2(0,10-1?M,10-2?M,10-3?M,10-4?M,10-5?M).The cell proliferation ability was measured at 450 nm absorbance?A?using CCK-8 kit,and cells were collected for determining the alkaline phosphatase activity.Results showed that:compared with control group,the cell viability of osteoblasts increased first and then decreased with increasing concentration of VK2.The 10-4?M,10-3?M,10-2?M of VK2 can significantly increase the viability of osteoblasts?P<0.05?.Compared with control group,the alkaline phosphatase activity in osteoblasts increased first and then decreased with increasing concentration of VK2.The 10-4?M and 10-3?M of VK2 can significantly increase the activity of alkaline phosphatase?P<0.05?.3.The alleviated effect of VK2 on the dexamethasone-induced injury of chicken embryo osteoblasts?1?The cells were treated with different levels of dexamethasone?100?M,80?M,60?M,40?M,20?M,0?M?,and the cell viability was measured by CCK8method.The results showed that the high concentration of dexamethasone could reduce the viability of osteoblasts,and the viability of osteoblasts decreased as the concentrations increased.Compared with the control group,the minimum concentration of dexamethasone that can reduce the viability of osteoblasts was 60?M?P<0.05?.?2?The cells were treated with different levels of VK2 and dexamethasone(0?M dexamethasone+0?M VK2,60?M dexamethasone+0?M VK2,60?M dexamethasone+10-5?M VK2,60?M dexamethasone+10-4?M VK2,60?M dexamethasone+10-3?M VK2,60?M dexamethasone+10-2?M VK2,60?M dexamethasone+10-1?M VK2),and the cell viability was measured by CCK8method.The results showed that:incubating osteoblasts with 60?M dexamethasone and different concentrations of VK2 together,the viability of osteoblasts with increasing concentrations of VK2 increased first,then decreased.Osteoblast viability was the highest in the dexamethasone-treated cells with 10-4?M of VK2?P<0.05?.4.In vivo feeding experiment of broilersA total of 360 day old Arbor Acre broilers were randomly assigned into three groups?0 mg/kg VK3 group,0.5 mg/kg VK3 group,4 mg/kg VK3 group?.Each treatment has 6 replicate pens and 20 chickens per pen.The control group was fed a basal diet?no extra vitamin K was added?,and the other two groups were fed with the basal diet supplemented with 0.5 mg/kg?NRC recommended level?and 4mg/kg of VK3,respectively.All chickens were pre-fed with the basal diet for 3days before formal trail,which lasted for 42 days.On days 21 and 42 of the trail,three chickens with similar weights were randomly selected,and blood was collected from the veins under the wing.Then,chickens were slaughtered to collect samples.The results showed that:4.1 Effect of VK3 on growth performance,leg disease and blood biochemical indexes of broiler chickensCompared with the vitamin K-deficient group,diet supplemented with 0.5mg/kg and 4 mg/kg of vitamin K3 significantly increased the average daily gain of chickens?d 21-42 and d 1-42??P<0.05?,reduced the ratio of feed to gain?d 1-21,d 21-42 and d 1-24??P<0.05?,but had no significant effect on average daily feed intake.Compared with the vitamin K-deficient group,the addition of 0.5 mg/kg or4 mg/kg of vitamin K3 significantly increased the serum calcium content at d 21?P<0.05?.Compared with the vitamin K-deficient group,the addition of 0.5 mg/kg of vitamin K significantly increased the serum calcium content at d 42?P<0.05?.Compared with the vitamin K-deficient group,the addition of 4 mg/kg of vitamin K3 significantly reduced the serum CREA level at d 21?P<0.05?.At d 42,vitamin K3 deficiency caused a significant increase in the incidence of leg disease?P<0.05?.4.2 Effects of VK3 on serum markers related with calcium and phosphorus metabolishResults showed that:compared with the vitamin K-deficient group,0.5 mg/kg vitamin K3 group could significantly increase the OCN level in serum at d 21?P<0.05?,reduce the BALP levels in serum?P>0.05?,and 4 mg/kg vitamin K3group could significantly reduce the BALP levels in serum?P<0.05?.Compared with the vitamin K-deficient group,0.5mg/kg vitamin K3 group could raise the OCN level in serum?P>0.05?,significantly reduce the serum BALP level?P<0.05?.Compared with the vitamin K-deficient group,4 mg/kg vitamin K3 group could significantly increase the level of serum OCN at d 42?P<0.05?,and reduce the BALP level in serum?P>0.05?.Compared to the 0 mg/kg of VK,dietary addition with 0.5 mg/kg of vitamin K3 could significantly reduce serum PTH levels?P<0.05?of chickens at d 21,4mg/kg of vitamin K3 reduced the plasma PTH level of chicken at d 21 and significantly increased the plasma levels of CT?P<0.05?.4.3 Effects of VK on bone quality indexes and tibia chemical compositionCompared with the vitamin K-deficient group,4 mg/kg vitamin K3 group increased calcium and the ration of calcium to phosphorus in the tibia at d 21?P<0.05?,and 0.5 mg/kg vitamin K3 group significantly increased the ash content in the tibia at d 21?P<0.05?.Compared with the vitamin K-deficient group,4mg/kg VK3 group significantly reduced the content of phosphate in tibia?P<0.05?,significantly increased the ration of calcium to phosphorus?P<0.05?.Compared with the vitamin K-deficient group,0.5 mg/kg and 4 mg/kg VK3 group significantly increased the ash content in tibia at d 42?P<0.05?.Compared with the vitamin K-deficient group,0.5 mg/kg VK3 group increased the strength of tibia at d 21?P>0.05?,and 4 mg/kg VK3 group significantly increased the strength of tibia at d 21?P<0.05?.Compared with the vitamin K-deficient group,0.5 mg/kg VK group increased the strength of tibia at d42?P<0.05?,4 mg/kg VK3 group significantly increased the strength of tibia at d21?P>0.05?.4.4 Effect of VK3 on the mRNA level of bone marker genes in tibiaCompared with vitamin K-deficient group,0.5mg/kg vitamin K3 group significantly increased the OCN and RUNX2 mRNA level at d 21?P<0.05?,had no significant effects on the ALPL mRNA level?P>0.05?.Compared with vitamin K3-deficient group,4 mg/kg vitamin K3 group significantly increased the ALPL,OCN and RUNX2 mRNA level?P<0.05?.Compared with vitamin K3deficient-group,0.5 or 4 mg/kg vitamin K3 group significantly increased the ALPL and RUNX2 mRNA levels at d 42?P<0.05?,and did not influence the OCN mRNA level?P>0.05?.In conclusion:incubation osteoblasts with vitamin K2 promoted the cell viability and the activity of alkaline phosphatase.In addition,the osteoblast inhibited by dexamethasone could be alleviated by vitamin K treatment.The leg disease occurred in broilers fed vitamin K-deficient diet.When chickens were fed with dietary supplementation of vitamin K3,bones got well.This might be due to that dietary addition with vitamin K3 promoted ALPL,OCN and RUNX2 gene expression,which stimulated the downstream signaling pathways and improved the bone formation and protection. |
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