Aquatic Research Models (Danio rerio and Nothobranchius furzeri): From Morphological Characterization to Stress Response Evaluation

Since the beginning of the twentieth century, the use of animal models has been pivotal in advancing various fields of research, ranging from basic to applied science. Fish species such as Danio rerio (zebrafish) and Nothobranchius furzeri (African turquoise killifish) have gained prominence due to their evolutionary significance, adaptability, and practical advantages in experimental studies. This thesis focuses on a comparative study of these two model organisms to deepen the understanding of their morphological and physiological characteristics. Choosing the appropriate animal model for a research project requires a thorough understanding of the species’ specific traits. Furthermore, in recent years, animal welfare has emerged as a critical focus in research, increasingly extending to aquatic animals, including fish. Enhancing our understanding of stress responses and applying the 3Rs principles (Replacement, Reduction, Refinement) is crucial for ensuring better welfare conditions for fish in research. The first chapter of this thesis provides a detailed morphological characterization of N. furzeri at two distinct larval stages, highlighting its rapid developmental processes and comparing these findings with existing zebrafish literature. This comparative analysis elucidates the unique and shared traits of these two species, emphasizing the importance of understanding species-specific adaptations when employing these models in research, as well as for proper husbandry and the application of relevant legislation. Chapter two delves into the chemical senses, specifically the olfactory and gustatory systems, in adult N. furzeri. A morphological analysis identifies N. furzeri as a microsmatic species, characterized by a less developed sense of smell. The olfactory epithelium is further analyzed using immunohistochemical markers. The gustatory system is examined through the distribution and count of taste buds in the skin, oral cavity, pharynx, gills, and esophagus, with cytotypes identified using different antibodies. Chapter three evaluates the stress response mechanisms of both D. rerio and N. furzeri under fasting conditions. The assessment includes cortisol levels in fin samples, oxidative product measurements in muscle tissue, and the analysis of Heat Shock Protein 70 (HSP70) through Western Blott and immunohistochemistry. Oxidative stress markers were also employed. The results indicate that a 96-hour fasting period does not result in significant changes in primary or secondary stress markers, suggesting that short-term fasting is not perceived as a major stressor. Additionally, the study explored alternative matrices for stress marker analysis to reduce the number of animals required, addressing the issue of small sample sizes in these species. Future research could investigate the physiological responses to a broader range of stressors, ultimately contributing to improved animal welfare in research settings. Finally, chapter four introduces glyoxal acid-free (GAF®) as a novel, non-toxic fixative for aquatic research models, with testing performed on zebrafish. The findings demonstrate that GAF® provides tissue preservation quality comparable to traditional toxic fixatives while enhancing safety and flexibility, particularly in environments lacking advanced safety infrastructure. Future studies should aim to validate the preservation of antigen epitopes through immunohistochemistry and explore GAF®’s applicability in marine species, potentially expanding its use in aquaculture research. In conclusion, this Ph.D. thesis contributes to the understanding of two significant fish model organisms by integrating morphological and physiological analyses. This work advances our knowledge of these species and promotes more refined and ethical research practices in aquatic model studies.