AN IN OVO TOXICOLOGICAL ASSESSMENT OF INDIVIDUAL AND COMBINED FUSARIUM MYCOTOXINS IN THE CHICKEN EMBRYO
Date
2019-03-11
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0001-9818-7675
Type
Thesis
Degree Level
Masters
Abstract
The increasing occurrence of Fusarium fungi and associated mycotoxins in cereal grains is a significant issue for global agriculture. The mycotoxin deoxynivalenol (DON) is the most prevalent feed contaminant worldwide and causes a variety of adverse effects in animals. While the individual toxicity of DON is a concern, exposure to multiple mycotoxins in feed is more common, necessitating information on the outcome of mixture exposures. However, characterizing the toxicity of DON and other mycotoxins has been difficult due to highly varied responses in long-term animal feeding trials. In addition, resources required for whole animal testing are only amplified in combinatorial mycotoxin studies given the large sample sizes and number of treatment groups required. The chicken embryo has been widely and successfully utilized as a non-animal alternative to evaluate the toxicity of environmental pollutants and could be used as a screening tool to evaluate mycotoxin mechanisms of action and mixture toxicity. The overall objective of this thesis research is to characterize the effects of DON (administered in ovo) alone and in combination with a commonly co-occurring mycotoxins, zearalenone (ZEA), to the late-term chicken embryo in order to determine whether an in ovo approach for conducting exposures to Fusarium mycotoxins could be used as a predictive tool for assessing the toxicity of Fusarium mycotoxins alone and in combination. The overall hypothesis is that responses of the late-term chicken embryo to single doses of Fusarium mycotoxins, alone or in combination, are similar to those reported in whole animal feeding trials with poultry.
In the first experiment, the effect of in ovo administration of DON was evaluated in terms of embryotoxicity, growth and development, pathological changes to tissue, and biochemical/molecular indicators of oxidative and immune stress. A single injection of purified DON was administered to the late-term chicken embryo (embryonic day 14, ED14) at five doses ranging from 0.0 – 5.0 μg DON/g egg weight. Eggs were opened on ED20 and embryos were evaluated for survivability and growth parameters. Tissues were sampled for subsequent analysis. At the highest dose, DON decreased embryo survivability and increased the absolute and relative weight of both liver and spleen. Hepatic bile stagnation and concurrent splenic inflammation were frequently detected among groups receiving 5.0 and 1.0 μg DON/g egg weight but were observed less often in the latter. A dose-dependent increase in granulopoiesis and lipid peroxidation (as measured by TBARS assay) were observed in the liver; however, mRNA expression of genes related to immune and oxidative stress were mostly unchanged. These results suggest that the chicken embryo responds to in ovo DON exposure with effects on immunity and oxidative stress that are supported by previous in vivo and in vitro findings. The in ovo approach developed and validated in the first experiment was then carried forward to a second experiment with the aim of characterizing the combined toxicity of DON and another mycotoxin, ZEA to the chicken embryo. ZEA was chosen for this experiment because the combination of DON and ZEA is considered to be the most prevalent mycotoxin mixture in North America and worldwide. Treatments included an untreated control group (CON), a vehicle-injected control group (20% DMSO), 0.5 and 2.5 μg DON/g egg weight, 0.5 and 2.5 μg ZEA/g egg weight, and a low and high combination treatment at 0.5 μg DON + 0.5 μg ZEA/g egg weight and 2.5 μg DON + 2.5 μg ZEA/g egg weight, respectively. The results demonstrated that interactive effects of DON and ZEA differed across endpoints and tended to vary from antagonistic at low doses to non-interactive or possibly potentiated at high doses. At low doses, DON and ZEA had antagonistic effects on liver weight as well as liver lipid peroxidation. At high doses, effects of DON and ZEA were mostly independent and effects of DON, specifically, were in line with our previous observations. At a combined, high dose of DON and ZEA there was evidence of possible potentiation with respect to embryo survivability, hepatic bile stagnation and splenic inflammation, and hepatic granulopoiesis. These results suggest the chicken in ovo model is useful for studying combinatorial mycotoxin toxicity; however, further research regarding ZEA-induced toxicity would improve response interpretation. Overall, the results presented in this thesis indicate that in ovo responses to Fusarium mycotoxins, alone and in combination, are supported by previous in vitro and in vivo findings. While in ovo mycotoxin exposures cannot replace in vivo experimentation, there is potential for the in ovo model to inform whole animal studies by identifying and prioritizing emerging mycotoxins and high-risk mycotoxin combinations for further in vivo assessment. In the future, the in ovo model could be used in a more practical application as a rapid-screening tool to assess the toxicity of mycotoxin grain extracts or to evaluate the efficacy of new mycotoxin mitigation techniques.
Description
Keywords
fusarium, mycotoxin, in ovo, chicken embryo, deoxynivalenol, zearalenone, oxidative stress
Citation
Degree
Master of Science (M.Sc.)
Department
Animal and Poultry Science
Program
Animal Science