Maturação ovocitária in vitro avaliada conforme sistemas de cultivo bidimensional e tridimensional

Abstract

The success of in vitro embryo production is directly linked to the quality of the oocytes and the conditions of in vitro culture. In the first stage of production called maturation, which refers to the in vitro culture of immature oocytes, the oocytes are selected and receive stimuli, follicular growth and composition from the medium in which they are inserted for the evolution of meiosis, involving nuclear and cytoplasmic changes essential for fertilization and subsequent embryonic development. However, productivity rates remain unchanged and further studies should be conducted in order to improve the quality of oocytes after the in vitro maturation phase. Thus, this study was conducted with two different experiments with the objective of: 1- Assessing the maturation of bovine oocytes with or without cumulus cells in three-dimensional in vitro culture systems using collagen matrix and comparing the traditional two-dimensional system; 2- Compare the methods of visual evaluation using an inverted microscope and fluorescence microscopy after the oocyte maturation process. The percentage of oocytes in the germinal vesicle was higher in those without cumulus cells. The oocytes with cumulus cells in a three-dimensional culture system showed a lower percentage of oocytes in metaphase I. Additionally, metaphase II rates were higher in those with cumulus cells regardless of the culture system. In addition, the percentage of ruptured oocytes was higher in the cumulus cells in 3D culture compared to the others. Different approaches were used to evaluate the oocyte chromatin post-maturation. In the evaluation by an inverted microscope, the parameters presence of the polar corpuscle and extrusion were evaluated. In general, the cumulus cells in 3D system showed a higher percentage of oocytes with polar body extrusion. The parameters analyzed germinal vesicle (fluorescence microscopy) versus absence of polar corpuscle (inverted microscope) and metaphase II (fluorescence microscopy) versus presence of polar corpuscle (inverted microscope) were compared between the approaches used: fluorescence microscopy versus inverted microscope. In general, the percentage of oocytes in the germinal vesicle (fluorescence microscopy) was lower compared to the proportion of oocytes observed with the absence of a polar corpuscle (inverted microscope). On the other hand, the percentage of oocytes in metaphase II (fluorescence microscopy) was higher than those identified with the presence of a polar corpuscle (inverted microscope). In summary, considering the results obtained through the analysis of fluorescence microscopy (elective as gold standard), the resumption of meiosis after the in vitro cultivation of oocytes with cumulus cells was similar between the 2D and 3D systems. Within the same evaluated system (2D or 3D), the absence of cumulus cells compromised the resumption of meiosis. In addition, oocytes matured in the 3D system showed a higher occurrence of damage in the pellucid zone.