Study On Toxic Effect And Metabolism Dynamics Of Four Mycotoxins In Laying Hens

Mycotoxins can cause cytotoxicity, immunotoxicity, genotoxicity, reproductive toxicity, carcinogenicity and mutagenicity on human and animal. Excessive or long-term low doses of mycotoxin exposure can transmit mycotoxins and their metabolites into animal, which then entry into human diet chain through meat, eggs, milk or other animal-derived food. The widely watched mycotoxins are deoxynivalenol, fumonisin, ochratoxin A, zearalenone and so on, and many countries or organizations have developed the relevant standards or guide limit values for these mycotoxins. However, the studies with respect to the toxic effect of these mycotoxins on laying hens are scarce. In addition, mycotoxin contamination in eggs has aroused widespread concern in mycotoxin wokers gradually, while the correlational studies are rarely reported due to insufficient monitoring methods. Therefore, it is essential to compare the differences of toxic effect on laying hens between different mycotoxins, develop methods for simultaneous determination of multiple mycotoxins in various matrices, carry out studies on mycotoxin metabolism in layers, and investigate the occurrence of mycotoxins in eggs, which can provide important references on toxicology and metabolism dynamics of mycotoxins, and is significant for ensuing egg safety and establishing the limit standards and relevant regulations. Contents and conclusions as follows:（1） This study was conducted to investigate the effect of respectively injecting the same dose（0.5 mg/kg.BW） of DON, FB₁, OTA and ZEA pure toxin by wing vein on growth performance, hematological indices and serum biochemical parameters of laying hens. The results showed: laying rate of all toxin treatment groups were decreased significantly, and the broken and soft egg ratio were also observably higher by injecting different toxins. Additionally, feed intake were markedly reduced in DON, OTA and ZEA groups. Compared with the control, PLT in different toxin treatment groups were significantly lessen, which was particularly evident in ZEA group. Otherwise, the parameters of WBC, LY, MO and GR in FB₁ treatment group were significantly increased compared with the control group. Furthermore, the ALP and GLUC content in serum of different toxin groups were lower than the control, but the indices of ALT, AST and TBIL were significantly higher than the control. By injecting DON, FB₁, OTA and ZEA pure toxin into wing vein of laying hens, production performance of laying hens were significantly descended, and the PLT were lessen as well as damage of the liver and kidney function was found in different groups, while ZEA was contributed to the most serious damage to production performance, coagulation system, liver and kidney function of laying hens.（2） This work established reliable and efficient LC-MS/MS methods for the analysis of 15 mycotoxins multi-residue in plasma, feces and eggs of layers. Various extraction procedures and clean-up procedures were first evaluated to ensure good recovery and minimize the matrix effect. Then, optimizing the chromatographic conditions on LC-MS/MS was used to enhance method sensitivity. Based on the experimental results, acetonitrile/formic acid（99/1, v/v） for plasma, with β-glucuronidase enzyme buffer for 2 h and acetonitrile/water solution containing 10% NaCl（84/16, v/v） for feces, with β-glucuronidase enzyme buffer for 6 h and acetonitrile/water solution containing 10 mM citric acid（84/16, v/v） for albumen and yolk, followed by oscillation and ultrasonic assisted extraction, then evaporation-reconstitution step for sample clean-up, also matrix-matched calibration curves were employed to ensure accurate quantification, which were selected as the optimal sample preparation procedures. According to the guidelines defined by Commission Decision 2002/657/EC and 401/2006/EC the performance characteristics（linearity, sensitivity, recovery, precision and specificity） of these developed methods were further validated in four sample matrices（plasma, feces, albumen and yolk）. The results showed that the inspection system established in this study had many advantages including low-cost of detection, high sensitivity and recovery rate, rapid analysis and so on, which could be applied to the analysis of large amounts of samples. According to established multi-mycotoxin detection methods, we investigated the mycotoxin contamination of albumen and yolk in Shanghai, which showed that the most common contamination toxins in eggs were zearalenones, and albumen were more susceptible to contamination than yolk.（3） This study was conducted to investigate the toxicokinetic in plasma, extent of excretion in feces and residual migration to albumen and yolk of DON, FB₁, OTA, ZEA and their metabolites in laying hens. By respectively wing-intravenous injecting the same dose（0.5 mg/kg.BW） of FB₁, DON, OTA and ZEA pure toxin for a single and last a week to laying hens, in which a single test was used to collect plasma and feces, and test for a week to collect eggs. The results showed: in plasma the times to peak concentration and the half-life of FB₁, DON, OTA and ZEA were 0.16, 0.25, 0.5, 0.5 h and 1.28, 2.26, 5.33, 12.04 h, respectively, and DON mainly metabolized 3-ADON, OTA partly metabolized OTα, ZEA metabolized β-ZOL and β-ZAL, also the elimination rate of metabolites were higher than their prototypes. In feces the total excretion and the times to complete excretion of FB₁, DON, OTA and ZEA were 68.27, 74.48, 51.49, 42.38% and 5, 5, 7, >7 d, respectively, also the total excretion rate of ZEA was significantly lower than other groups, and DON with DOM, OTA with OTα and ZEA with β-ZOL, β-ZAL excreted. The residual peak concentrations of FB₁, DON, OTA and ZEA in albumen and yolk were 18.24, 15.26, 23.63, 27.64 μg/kg and 6.06, 0, 0, 11.97 μg/kg, respectively, and the residual times to completely eliminate were 11, 11, 14, >14 d, also the residual kinds and concentrations of these toxins in albumen were significantly higher than yolk. Therefore, in laying hens FB₁ and DON had higher elimination speed and excretion efficiency than OTA and ZEA, which meaned that OTA and ZEA had longer toxic effect on laying hens, and albumen were more susceptible to migration and vestigital of mycotoxins than yolk. Besides, ZEA and its metabolites had the most serious accumulation effect and the slowest speed of remove vestigital in eggs, OTA followed, then FB₁ and DON had lower residual amount.