The Coagulation Disorder Of Yellow Feather Broiler Induced By Sodium Dehydroacetate And The Antagonistic Effect Of The VK

Sodium dehydroacetate（DHA-S）is widely used in feed,food,cosmetics,medical chemical and other fields as an antibacterial and antiseptic additive.However,studies in our and other laboratories have shown that DHA-S can cause coagulation disorders in mammals such as rats,mice and rabbits,prolonged prothrombin time（PT）,and activated partial thromboplastin time（APTT）;decreased serum vitamin K（VK）,coagulation factor IX（FIX）content;and inhibited expression of liver vitamin K epoxide reductase complex 1（VKORC1）,and vitamin K epoxide reductase complex-1 like protein 1（VKORC1L1）.The physiological structure and coagulation system of poultry are different from those of mammals,and poultry are sensitive to VK deficiency.There are few reports on effects of DHA-S used as a feed additive on the growth and coagulation function of chickens.This study,through feeding experiments in chickens and in vitro culture of chicken LMH cells,intends to understand the effect of DHA-S on the growth and coagulation function of broilers and to explore the mechanism of DHA-S affecting coagulation function.The research results can provide a theoretical basis for the rational and safe use of DHA-S as a feed additive in clinical and the risk of VK deficiency in chickens,and have positive significance for the prevention of potential coagulation disorders of DHA-S.During the experiment of the effect of DHA-S on the growth and coagulation function of yellow-feather broilers,1 day-old yellow feather broilers were given 100,200 and 400 mg/kg feed doses of DHA-S respectively.Blood was collected at different times to measure PT,APTT and plasma recalcification time（PRT）;ELISA method was used to measure serum contents of VK and FIX;the blood drug concentration was determined by HPLC;the feed intake,weight gain,blood biochemical indicators,liver function indicators,organ coefficients,etc.were detected,and the histopathological analysis of the main organs was carried out.The results showed that 200 and 400 mg/kg DHA-S could significantly prolong the coagulation time of PT,APTT and PRT in yellow feather broilers（P<0.05）,and significantly reduce the contents of VK and FIX in serum（P<0.05）.After administration with different doses of DHA-S for 2 weeks,the concentration of DHA-S in the blood tends to be stable,and the blood concentration is positively correlated with the dose.DHA-S had no significant effect on chicken weight gain,feed consumption,liver function,and main blood biochemical indicators.For the experiment of the effect of DHA-S on the growth of LMH cells and related factors of blood coagulation,the cells were treated with 0.1～10.0 mmol/L DHA-S for 24 h,cell viability was determined by MTT method;the contents of VK and FIX in the cells were determined by ELISA;the expressions of VKORC1 and VKORC1L1 proteins in the cells were determined by Western blot.The results showed that 5.0 and 10.0 mmol/L DHA-S could significantly inhibit cell viability（P<0.001）;significantly reduce the contents of VK and FIX in cells and supernatant（P<0.05）;significantly decreased the protein expression of VKORC1 and VKORC1L1 in cells was（P<0.001）.In the study of the antagonistic effect of VK on coagulation disorders caused by DHA-S,① broilers were administered with 100～400 mg/kg DHA-S to for 2 weeks,and then divided into DHA-S group,DHA-S+VK group,and VK group respectively.Chickens were injected intramuscularly with 0.5 mL of VK.24 h and 48 h after injection,blood was collected to determine coagulation indexes and the contents of VK and FIX in serum.② LMH cells were co-treated with 2.0～8.0 μmol/L VK and 5.0 mmol/L DHA-S for 24 h,the cell viability was measured by MTT method;the contents of VK and FIX in the cells were measured by ELISA.The results showed that ① the PT,APTT and PRT values of broilers were significantly decreased after VK injection following with different doses of DHA-S administration,while the contents of VK and FIX were significantly increased,and the difference was significant at 48 h after VK injection（P<0.05）.②2.0～8.0 μmol/L VK could significantly alleviate DHA-S decreased cell viability in LMH cells;8.0 μmol/L VK could completely antagonize the decrease of cell viability caused by 5.0 mmol/L DHA-S（P>0.05）,which was comparable to the control group.Compared with the control group,cotreatment with 4.0,8.0 μmol/L VK and 5.0 mmol/L DHA-S,there was no significant difference in intracellular VK and FIX content（P>0.05）.In conclusion,100～400 mg/kg DHA-S can inhibit the expression of VKORC1 and VKORC1L1 in the liver,prolong the coagulation time,reduce the contents of VK and FIX,and lead to coagulation dysfunction in broilers.No other adverse effect was observed.DHAS can reduce the content of VK and FIX,and inhibit the expression of VKORC1 and VKORC1L1 in LMH cells.VK has a significant antagonistic effect on the coagulation dysfunction caused by DHA-S in broilers;it has a protective effect on the reduction of coagulation factors in LMH cells caused by DHA-S.It suggests that VK can significantly alleviate the effect of DHA-S on liver coagulation factors at the cellular level,and can effectively antagonize the coagulation disorder caused by DHA-S in chickens.