Effects Of Sodium Dehydroacetate On Coagulation Function And VKOR Expression In Broilers

Sodium dehydroacetate(DHA-S)is a common preservative in food and feed production,which has a good inhibitory effect on bacteria,yeasts,and molds that cause spoilage.The data reported that DHA-S can prolong the coagulation indexes such as prothrombin time(PT)and activated partial thromboplastin time(APTT),cause bleeding tendency in different organs of animals when was given to rats and rabbits in larger doses.The disturbance of blood coagulation induced by DHA-S in rats is related to the inhibition of VK epoxide reductase(VKOR).The VK 2,3-epoxide reductase complex 1(VKORC1)and VK 2,3-epoxide reductase complex 1-like protein 1(VKORC1L1)are two subtypes of VKOR enzyme.There was different distrubution of VKORC1 and VKORC1L1,which VKORC1 was majority in rat liver and VKORC1L1 was mainly in extrahepatic tissues.No investigation about the possible effect of DHA-S as a feed mold inhibitor on the coagulation function of poultry were reported so far.In this paper,oral administration of DHA-S was given to broilers,the content of coagulation indexes PT,APTT,serum VK and FIX were detected,and the distribution of DHA-S in blood and tissues was determined by HPLC method to evaluate the effect of DHA-S on broiler coagulation function.Western-Blot and immunohistochemical methods were used to analyze the expression levels of VKORC1 and VKORC1L1 in different tissues to explore the VKOR mechanism of the effect of DHA-S on the coagulation function of broilers.It will be useful for data accumulation to the rational use and application safety evaluation of DHA-S used as animal feed additive.Broilers were given 100,150,and 200 mg/kg b.w DHA-S orally,and the PT and APTT values at different time points were detected by coagulometer,and the content of PT,VK,and FIX in serum was detected by ELISA method.HPLC was used to detect the content of DHA-S concentration in blood,liver,lung and other tissues,Western Blot and immunohistochemical methods were used to analyze the expression levels of VKORC1 and VKORC1L1 in the tissues.The correlation between DHA-S concentration in blood,PT,APTT,and the expression of VKORC1 and VKORC1L1 in tissues were analyzed.Results showed that the PT and APTT values in 100 mg/kg DHA-S was appeared changed but no significant difference compared to control.When 150 mg/kg DHA-S was given for a week,the PT and APTT values were significantly prolonged at different time points of 4 h,8 h,5 d,and 7 d(P<0.05),with about 1.10?1.30 times in PT,1.15?1.35 times in APTT higher than that of the control group.The serum VK content were all decreased significantly at different time points(P<0.05).The serum FIX were decreased significantly at 4 h and 8 h(P<0.05).When 200 mg/kg DHA-S was given for three consecutive days,the PT and APTT values were significantly prolonged at 4 h,8 h,16 h,24 h,48 h,and 72 h(P<0.05).The VK content and FIX level in the serum were significantly reduced to varying degrees(P<0.05).HPLC was used to detect the content of DHA-S in chicken serum and tissues.The results showed that serum DHA-S concentration at 4 h and 8 h were significantly higher than at 16 h and 24 h after 150 mg/kg or 200 mg/kg administration(P<0.05).The peak concentration of DHA-S in different tissues appeared at 8 h after administration.The drug concentration in the liver and lungs of hens were significantly higher than that of cocks at the same time(P<0.05).Western Blot analysis showed that the expression of VKORC1 and VKORC1L1 in broiler liver and lungs were all significantly reduced,about half of the control group after DHA-S 150 mg/kg single administration or 200 mg/kg continuous administration(P<0.05).VKORC1L1 protein in ovarian tissue also decreased significantly(P<0.05),about 1/2 of the control group.Immunohistochemical results showed that the expression of VKORC1 in chicken liver,lung,ovary and testis tissues were significantly reduced compared with the control group.Correlation analysis results showed that the serum DHA-S concentration was positively correlated with PT and APTT,and the correlation coefficient in cocks with 0.82 in PT and 0.86 in APTT were significant higher than that of hens 0.69 in PT and 0.31 in APTT(P<0.05).Serum DHA-S concentration was negatively correlated with VKORC1 and VKORC1L1 levels in tissues,higher correlation coefficient in liver than in lung.In summary,DHA-S of higher doses was found can prolong coagulation index PT and APTT values of broilers,and decrease the content of VK and FIX in serum,which appeared the risk of causing coagulation disorders in broilers.The concentration of DHA-S in hen tissues is higher than that in males.The obvious inhibition of DHA-S on VKORC1 and VKORC1L1 in chicken tissues is one of the main causes of coagulation dysfunction in broilers caused by DHA-S.The research results can provide a theoretical basis for the reasonable clinical application of DHA-S and its mechanism of abnormal coagulation function in broilers.