Effectiveness And Safety Of Cyadox In Laying Hens

Quinoxalines are broad-spectrum antimicrobial agents with growth-promoting activity,and have been used extensively in food animals since 1970s.Carbadox and olaquindox are the early members,which have been limited to use in food animals in some countries for their genetic or potential toxicities.Cyadox is a new candidate of quinoxalines.Some studies show that cyadox can promote the growth and improve the feed conversion in swine,sheep and fish with much lower toxicity than olaquindox. Hence,it is hopeful that cyadox would be developed as a replacer of olaquindox and carbadox with more safety and excellent effectiveness in growth promotion and antibacterial activity.But it has not been found any report concerning the effectiveness and safety studies of cyadox in laying hens.1.In vitro antibacterial activity of cyadox on layer bacteria.Determination the in vitro antibacterial activity of cyadox,aureomycin and zinc bacitracin against Salmonella,Escherichia coli,Pasteurella multocida.S.aureus by microdilution.Cyadox was active both on gram-positive bacteria and gram- negative bacteria,and more active on Escherichia coli and S almonella pullorum than the others. The MIC of these was 16 and 4μg/mL respectively,which were lower than that of aureomycin and zinc bacitracin.The antibacterial activity of cyadox against Pasteurella multocida was weaker than that of aureomycin.But the antibacterial activity of cyadox against S.aureus was the same as that of aureomycin.The results indicated that cyadox could be used as an antibacterial agent clinically.2.The effects of cyadox on performance,egg quality and characters of physiology and biochemistry in layers.1260 layers were randomly allotted to 7 treatments with 3 replicates.The treatments were:one group fed the basal diet without any antimicrobial,one group fed the basal diet containing 50mg/kg zinc bacitracin,and the other five groups fed the basal diet containing 25,50,100,200,500mg/kg cyadox,respectively.The animals at the start of the 7-day adaptation period were fed a basal diet containing no antibiotics.Records were made of the quantity of egg,egg weight,cracked and soft eggs,and then to compute the egg production rate,mean egg weight,daily egg mass,weekly feed intake,feed conversion and mortality.30 eggs from each treatment were used to evaluate egg-quality, which performed three the first before the experimenl,the second after 7 weeks of treatment and the last at the end of the experiment.Egg quality was based on determination of egg weight,egg specific gravity,egg-shape index,shell thickness,and half unit.The serum was collected on weeks 0 and 12,to determine the biochemistry characters.The effects of cyadox on performance in layers:the results showed that cyadox can improve laying performance.Egg production rate in groups treated with cyadox 100mg/kg was higher compared with the control group and increased 2.43%(P＞0.05). Egg production rate in groups treated with cyadox 100mg/kg was higher compared with the groups of 500mg/kg cyadox and increased 5.71%(P＜0.05).Egg weight and egg mass in groups of 100mg/kg cyadox was increased by 1.64%and 7.16%compared with the group of 500mg/kg cyadox,respectively(P＜0.05).Feed consumption in 500mg/kg cyadox was depressed by 6.73%compared with the control group(P＜0.01).Feed conversion in groups treated with cyadox(50 and 100mg/kg) was depressed by 7.07% and 8.84%compared with the control group,and Feed conversion in 100mg/kg cyadox was depressed by 1.11%compared with the the group of Zinc bacitracin(P＞0.05).All of these indicated that 50 and 100mg/kg cyadox can improve egg production rate,egg weight,egg mass and depressed the feed consumption and feed conversion.The effect of cyadox with dose of 100mg/kg was equal to Zinc Bacitracin.The effects of cyadox on egg quality in layers:in the middle of the experiment (39wk),two dose of cyadox(50 and 100mg/kg) can largely increase egg shell thickness (P＜0.01).Half unit in group treated with cyadox(100mg/kg) was the highest,and the control group was the lowest.Differences between the two groups were very significant (p＜0.01).At the end of the experiment(44wk),egg shell thickness and half unit in group treated with cyadox(100mg/kg)was also the highest,and the control group was the lowest, but differences between the two groups were not significant(p＞0.05).There were no obviously differences among the groups in egg weight,egg specific gravity and egg-shape index(p＞0.05).All of these indicated that cyadox with dose of 50mg/kg to 100mg/kg can improve egg quality.The effects of cyadox on characters of physiology and biochemistry in layers:no obvious effects of cyadox on relative characters of physiology was observed in all treatment groups cxcept red blood cell and hematocrit.The results showed that red blood cell and hematocrit in group treated with cyadox 100mg/kg was significantly higher compared with the group of 500mg/kg cyadox(P＜0.05),and higher compared with the control group(P＞0.05).No obvious effect of cyadox on relative characters of biochemistry,was observed in all treatment groups except glucose and Na⁺.The results showed that glucose in group treated with cyadox 100mg/kg was extremely higher compared with the group of 500mg/kg cyadox(P＜0.01),and significantly higher compared with the control group (P＜0.05).Na⁺ in group treated with cyadox 500mg/kg was significantly lower compared with the control group(P＜0.05).However,all the relative characters of physiology and biochemistry in any group were normal.3.Residue depletion of eyadox in layers.Methods of quantitative analysis:according to the analytical methodology in biological samples,HPLC method has been developed for the detection of cyadox and its main metabolites in eggs.The first method was the extraction of cyadox and desoxy-cyadox.Egg samples were subjected to acid hydrolysis with HCl,followed by extraction with acetonitrile/chloroform(1:4,v/v) and degrease with n-hexane.The column was an Agilent TC-C₁₈ column(250mm×4.6mm i.d.,5μm particle sizes).Cyadox and desoxy-cyadox were separated by water and acetonitrile with a gradient elution condition. The detailed gradient elution program was as follows:time(min),water(%),acetonitrile (%):0,80,20;8,80,20;12,75,25;25,75,25.The results showed that the observed limit of determination for cyadox and desoxy-cyadox were 40ug/kg.The mean recoveries of cyadox and desoxy-cyadox,spiked at levels of 40,80,160μg/kg,were from 68.32%to 83.28%.The inter-day relative standard deviation for two compounds were 5.64%-10.58%.The second method was the extraction of quinoxaline-2-carboxylic acid.Egg samples were subjected to acid hydrolysis with 10%metaphosphoric in 20%methanol, followed by extraction with ethyl acetate and phosphate buffer and Oasis MAX solid-phase extraction sequently clean-up.The column was an Agilent HC-C₁₈ column (250mm×4.6mm i.d.,5μm particle sizes).The mobile phase was prepared from acetonitrile/water containing 1%formic acid(20:80,v/v),and the detection was at 320nm. The flow rate was 1.0mL/min.The results showed that the observed limit of determination for quinoxaline-2-carboxylic acid was 4ug/kg.The mean recoveries of quinoxaline-2- carboxylic acid,spiked at levels of 4,8,16μg/kg,were from 69.14 to 72.78%.The inter-day relative standard deviations were lower than 8.96%.The experiment of Residue depletion:the method described above was used to determine the residues of cyadox,desoxy-cyadox,and in eggs,and only quinoxaline-2-carboxylic acid in muscle and liver.At the end of phase,20 eggs from the group of 100mg/kg were daily collected to determine the residues of the parent drug, desoxy-cyadox and quinoxaline-2-carboxylic acid.30 layers were killed at the end of the experiment to determine the residues of quinoxaline-2-carboxylic acid in muscle and liver. The result indicated that there were no residues in eggs and muscle.After a withdrawal time of 12h,the concentration of quinoxaline-2-carboxylic acid in liver was very high all the same,but it was below the LOQ(10μg/kg) after a withdrawal time of 6d.In a word, cyadox had low residue after long term administration,which indicated that cyadox was safe enough to layers.So far,no MRLs of quinoxaline-2-carboxylic acid has been established in layers,based on the toxicology results,a temporary MRL of 0.9μg/g was established.Since the concentration of quinoxaline-2-carboxylic acid in liver after a withdrawal period of 12h was much lower than 0.9μg/g,it can be concluded that no withdrawal time was needed for cyadox in layers.In conclusion,this study opened out the good application foreground of cyadox in layers.The results confirmed the maximum safe dose and reasonable application dose of cyadox in layers,and clarified the residue depletion characteristics of cyadox in layers. This study also established the withdrawal time of cyadox in layers.Moreover,feasible analytical methods of cyadox and its metabolites were developed to control the redsidues of cyadox.These results would supply the important datas for the effectiveness and safety evaluation of cyadox in layers,and give scientific guidance for the reasonable and safe use of cyadox in layers.In short,cyadox was a safe for layers with excellent growth promotion effect and antimicrobial activity.