INTRODUCTION
The use of scanning electron microscopy (SEM) allows precise identification of the surface of structures through the generation of three-dimensional images.1 This technique is based on a beam of electrons, produced by an electron source gun that is generated by a filament or crystal that focuses on a region of the sample. Then, this beam is transmitted to a cathode screen, and the generated image is the result of an interaction that occurs between beam-sample.2 However, to perform the technique, several aspects must be considered, such as: electrical conductivity, hardness, and porosity of the sample to be analyzed.3For example, samples that have low conductivity need to undergo a metallic coating, usually a gold film is used. 3
SEM analyses are used to reveal microstructures, showing the size, structure, and shape of particulate or fractured surfaces, such as: polymers, proteins, seeds, inorganic, and organic compounds.1,4-5 In this regard, the association of the cryofracture technique adds to the characteristics that can be observed under the microscope. This protocol allows for an analysis with more detailed observations, since the samples have their internal structures exposed, promoting a finer resolution of structures, and using the depth of field created by SEM.6
As evidenced by Riehl7 in a study with teleosts, the pattern found on the surface of the oocyte can be used as one of the criteria for identification and differentiation of species. Macchiarelli et al.5 also report that this technique allows the morphofunctional classification by understanding the vascular plexuses.
It is clear, then, that the analysis of oocyte structures involves the use of high precision techniques, as proposed in this study. It is worth mentioning that in teleosts, as in other vertebrates, the oocyte development in a mature egg is a regulated process under the influence of environmental and endocrine factors.8 Thus, the clearer the understanding of the morphological characteristics of the oocytes, the easier and more complete the understanding of the dynamics of oogenesis will be.9 The understanding of cellular growth dynamics is used also to describe the reproductive cycle, allowing the recognition of the reproduction period and the morphological modifications that occur.10
SEM can therefore be used as a complementary tool to light microscopy, as it provides more details.4 However, until now, no studies have been found that used SEM associated with cryofracture for the description and comparison of fish oocytes, so it is not possible to estimate its real application in these models. Thus, the protocol and results of SEM associated with cryofracture in Astyanax lacustrisovaries are presented here in order to characterize the external and internal structure of the oocytes.