The Field Trial Of Cyadox In Laying Ducks

Cyadox (Cyx),(2-formylquinoxaline)-N1, N4-dioxide cyanoacetyl-hydrazone, is a novel antimicrobial growth-promoter of the quinoxalines. Compared with carbadox and olaquindox, well-known members of the quinoxalines, which were banned or limited to be used in food animals for toxicities, cyadox was more effective and safer in promoting the growth of animals. In the last years, lots of researches had been performed to evaluate the effectiveness and safety of cyadox in swine, chicken and fish. The dose of studies demonstrated that cyadox was outstanding active to kill pathogenic bacteria, and could improve the growth and feed conversion efficiency with lower toxicity and side effects. This indicated that cyadox could be a promising candidate of quinoxalines to animal husbandry compared with its congeners. But it has not been found any reports concerning the effectiveness and safety studies of cyadox in laying ducks. For practice use and food safety concerns, it is necessary to develop the field trial of cyadox in laying ducks.1. Antibacterial activity of cyadox on duck bacteria in vitro.The in vitro antibacterial activities of cyadox on5species13strains pathogeric bacteria of ducks (Escherichia coli, Salmonella, Pasteurella multocida, Staphylococcus aureus, Riemerella anantipestifer) were tested by Broth microdilution method, which recommended by Clinical and Laboratory Standards Institute (CLSI). The results showed that cyadox was active on duck bacteria, and more active on Pasteurella multocida and Staphylococcus aureus than the others. The MIC of these were32and4～8μg/mL, but the others were64～128μg/mL. The antibacterial activity of cyadox against Pasteurella multocida was a little weaker than that of aureomycin, but they were similar on the other bacteria(Escherichia coli, Salmonella, Staphylococcus aureus, Riemerella anantipestifer). At the time, the antibacterial activity of cyadox against all the bacteria was stronger than that of zinc bacitracin and olaquindox. The results indicated that cyadox could be used as an antibacterial agent clinically.2. The long-term trial of cyadox in laying ducks.2160laying ducks were randomly grouped to4treatments with3replicates. One group fed the basal diet without any antimicrobial, which was regarded as the control group. Then the other three groups were cyadox groups, which fed the basal diet containing50,100,200mg/kg cyadox, respectively. In the whole trial, we had recorded egg quantity, egg weight, cracked and soft eggs everday and recorded feed intake, feed conversion and mortality weekly, and computed the egg production rate, mean egg weight and daily egg mass. At the middle and the end of the experiment,30eggs from each treatment were used to evaluate egg qualities, which including egg specific gravity, egg-shape index, shell thickness, yolk rate, yolk color and haugh unit. At the middle and the end of the experiment,10ducks from each treatment were used to determine the physiology and biochemistry characters.The effects of cyadox on performance:The results showed that cyadox can availably improve performance in laying ducks. Laying rate in the group treated with cyadox100mg/kg was the highest, and increased2.56%(P<0.05) compared with the control group. The second was50mg/kg. Egg mass and egg weight in the group treated with cyadox100mg/kg were increased3.31%and0.76%(P>0.05) compared with the control group. Feed consumption in the group treated with cyadox100mg/kg was depressed by3.15%(P>0.05) compared with the control group. All of these indicated that cyadox with the dose of50-200mg/kg can increase laying rate, egg mass, egg weight and decrease the feed conversion.The effects of cyadox on egg qualities:The results showed that cyadox can availably improve egg qualities in laying ducks. In the middle of the experiment (44wk), the group treated with cyadox100mg/kg can largely increase egg weight, haugh unit, yolk color and shell thickness, and increase9.10%(P<0.01),13.34%(P<0.01),6.89%(P>0.05) and8.70%(P<0.01) by turns compared with the control group. At the end of the experiment (50wk), the group treated with cyadox100mg/kg also can largely increase egg weight, haugh unit, yolk color and shell thickness, and increase6.84%(P<0.01),1.73%(P>0.05),21.72%(P<0.01) and9.33%(P<0.01) by turns compared with the control group. There were no obviously differences among the groups in egg-shape index, yolk ratio and egg specific gravity (P>0.05). All of these indicated that cyadox with the dose of50-200mg/kg can improve egg qualities.The effects of cyadox on characters of physiology and biochemistry:There was no obvious effect of cyadox on relative characters of physiology and biochemistry in all treatment groups, and they were all in the normal physio-scope. In the middle of the experiment, mean corpuscular hemoglobin of the groups treated with cyadox were higher than of the control group, but there was no significant difference. In the end of the experiment, red blood cell, hematocrit and K+of the groups treated with cyadox were higher than of the control group, as well, there was no significant difference. In the middle and the end of the experiment, glucose, alanine amiotransferase and aspartate aminotransferase of the groups treated with cyadox were higher than that of the control group, also, there was no significant difference. Anyway, all the relative characters of physiology and biochemistry in any group were normal.The effects of cyadox on species and quantity of common pathogenic bacteria: The results showed that cyadox can decrease species and quantity of common pathogenic bacteria in laying ducks. For Escherichia coli in cecum, the groups of cyadox can largely decrease the quantity of Escherichia coli, and the groups treated with cyadox50,100and200mg/kg were decreased9.1%(P>0.05),12.5%(P<0.01) and11.2%(P<0.05) compared with the control group. For the other bacteria(Salmonella, Pasteurella multocida, Staphylococcus aureus and Riemerella anantipestifer), cyadox can inhibite them and showed a time-and dose-dependent manner. When the dose of cyadox was200mg/kg, no Salmonella, Pasteurella multocida, Staphylococcus aureus and Riemerella anantipestifer had been isolated.3. Residue depletion of cyadox in laying ducks.Methods of quantitative analysis:Two methods of high-performance liquid chromatography with UV have been established for the simultaneous quantitative determination of Cyx and its six major metabolites (Cyl, Cy2, Cy4, Cy6, Cy9and Cyl2) in eggs and edible tissues of ducks. For eggs (egg white and egg yolk), samples were extracted with2%metaphosphoric acid in methanol/acetonitrile/water (40:20:40, v/v/v), followed by liquid-liquid extraction and HLB solid-phase extraction clean-up. For tissues (liver, kidney, muscle and fat), samples were extracted with1%metaphosphoric acid in methanol/acetonitrile/water (50:10:40, v/v/v), followed by HLB solid-phase extraction clean-up and liquid-liquid extraction. Analysis was performed on a ZORBAX SB C18liquid chromatography column by UV detection with a gradient program of wavelength. For eggs, the limits quantification (LOQ) for Cyx, Cyl, Cy2, Cy6, Cy12were16μg/mL in egg white, and Cy9and Cy4were8μg/mL and32μg/mL, respectively; the LOQ for Cyx, Cy1, Cy2, Cy6, Cy9, Cy12were16μg/mL, and Cy4was32μg/mL in egg yolk. In the edible tissues, the LOQ for Cy1, Cy2, Cy6, Cy12were20μg/mL in liver, kidney, muscle. fat, Cy9was10μg/mL in kidney, muscle, fat and20μg/mL in liver, Cyx was10μg/mL in muscle and20μg/mL in liver, kidney, fat. The recoveries of seven analytes in egg white and yolk, spiked at levels of8μg/mL～128μg/mL, were64.9%～80.1%, with inter-day relative standard deviation (RSD) less than9.3%. The recoveries in edible tissues, spiked at levels of10μg/mL～160μg/mL, were63.4%～79.8%, with inter-day RSD less than10.5%. The developed methods, which were simple, fast and economic, could be applied to the monitoring for the possible residues of cyadox and its metabolites in eggs and edible tissues of ducks.The experiment of residue depletion:Cyadox was administrated to laying ducks in medicated feed at the level of100mg/kg for consective20days, the HPLC methods were used to determine the residues of cyadox and its six major metabolites in eggs (egg white and egg yolk) and tissues (liver, kidney, muscle and fat). For eggs, after a withdrawal time of Od, only Cy4could be detected, and it eliminated below LOQ after a withdrawal time of Id in egg white and egg yolk. For tissues, after a withdrawal time of6h, cyadox and its five major metabolites (Cy1, Cy2, Cy4, Cy6, Cy12) were detected in liver, and Cyx, Cy1, Cy4, Cy6, Cy12in kidney, and Cyx, Cyl, Cy4in muscle, and Cy4in fat. After a withdrawal time of5d, Cyx, Cy1, Cy2, Cy4, Cy6and Cy12in edible tissues were all eliminated below LOQ, except Cyl in kidney. Therefore, Cyl was the most persistent metabolite in tissues and was more persistent in kidney than the other tissues. For the reason, kidney was inferred to the target tissue, and Cyl was the marker residue of cyadox in laying ducks.In conclusion, this paper studied the effectiveness and safety on laying ducks for field trial. This study confirmed the reasonable application dose of cyadox in laying ducks, and developed the residual analytical methods of cyadox and its metabolites in duck eggs and duck tissues. This study also clarified the residue depletion characteristics of cyadox in laying ducks and established the withdrawal time. These results gave the scientific guidance for the reasonable and safe use of cyadox in laying ducks. In short, it is promising for cyadox to have a good application foreground in laying ducks with excellent growth promotion effect and antimicrobial activity.