Experimental study of the embryonic development of common toad (Bufo bufo): methodological aspects

Embryos and larvae of many amphibian species are widely used as experimental models in the areas of developmental biology, toxicology and fundamental medicine. However, there are almost no experimental approaches considering the biology of particular species. Moreover, experimental conditions are not standardized between different laboratories, and there are no examples of making efforts to minimize an influence of the researcher’s personality on the results of experiments. The worse thing is that many experimental methods designed for particular species are applied for other amphibians without an adaptation. In the presented paper, the results of long-term experimental investigations of environmental factors influencing embryonic development of the common toad Bufo bufo are summarized. These investigations are an example of establishing the methods and approaches considering the species biology, allowing minimizing heterogeneity of an influence of stochastic factors and standardizing experimental conditions. For this species, it is recommended to maintain the temperature at 15°C in the course of all experiments, to perform all measurements with 8 hr intervals, and to perform each test in at least two replicas, which differ in the initial density of embryos (clutch fragments containing 30 or 120 embryos per standardized aquarium). When designing experiments, it is suggested to consider not only volumetric density of embryos (the number of embryos per unit of volume), but also their surface density (the number per unit of area) and linear density (the number per unit of roe cord length). It was experimentally shown that it is necessary to keep the interactions between the researcher and the object at a minimal level, as all kinds of researcher actions, including removal of death embryos, severely affect the results of experiments.

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