Cultivo in vitro de folículos pré-antrais bovinos associado à nanopartículas e levitação magnética

Abstract

The aim of this study was to evaluate the development of secondary follicles in the in vitro culture on a three-dimensional system of nanoparticles (gold, iron oxide and poly-L-lysine) associated with magnetic levitation at three concentrations (50, 100 and 200 μL/mL) compared to 2D in vitro culture (control, traditional) for 16 days. Sixty bovine ovaries were collected and the micro dissection of the secondary preantral follicles (200 μm to 400 μm in diameter) was done manually from the fragments of the ovarian cortex. Thereafter, the isolated follicles were individually transferred to micro tubes containing culture medium. For 3D treatments, the nanoparticles were added according to the proposed concentrations. After 12 hours of incubation, the follicles were transferred to 24-well plates with culture medium and cultured for 16 days. At the end of the culture, the oocytes were in vitro matured for 22-24 hours. A total of 213 preantral follicles were selected for in vitro culture and randomly assigned to the treatments. The 200 μL/mL treatment showed a reduction (P < 0.05) in follicular viability only from D12 and at the end of the culture (D16), the highest proportion of viable follicles (P < 0.05) was observed in this group. The treatments with the highest concentrations of nanoparticles had the lowest rates of oocyte extrusion and higher proportion of antrum formation. In addition, within the slow-growing category, a superior percentage of follicles (P < 0.05) was observed in the 200 μL/mL group. The follicles of the control group were 6 times more likely (P < 0.05) of follicular degeneration and/or extrusion when compared to 200 μL/mL group. Additionally, the oocyte viability rate in in vitro maturation was higher (P < 0.05) in the treatment of 200 μL/mL. Furthermore, only the oocytes developed in the 3D culture reached the MI phase after the resumption of meiosis. Finally, it was concluded that the three-dimensional culture system with nanoparticles associated with magnetic levitation promoted the in vitro development of bovine secondary follicles and the concentration of 200 μL/mL obtained the highest proportion of viable follicles with the best rates of antrum formation and maintenance.