Avaliação do efeito antioxidante do óleo essencial das folhas de Croton Argyrophyllus Kunth durante o cultivo in vitro de folículos pré-antrais inclusos no tecido ovariano bovino

Abstract

The aim of this study was to evaluate the antioxidant effect of Croton argyrophyllus Kunth Essential Oil (OECA) in the in vitro culture of preantral follicles included in bovine ovarian tissue, considering its influence on follicle activation and development, morphological integrity of the follicles, stromal cell density and configuration of the extracellular matrix (ECM). To this end, the antioxidant capacity of OECA was determined by scanning for the 2,2-diphenyl- 1-picrylhydrazyl radical (DPPH) and the 2,2'-azinobis (3-ethylbenzoathiazoline-6-sulfonic acid) radical (ABTS). In the laboratory, fragments of ovarian cortex (3x3x1 mm) were fixed in 10% paraformaldehyde (uncultured control) or cultured in vitro in 500 µL of α-MEM control medium alone, consisting of BSA at 1.25 mg/mL, glutamine at 2 mM, penicillin/streptomycin 100 μL, hypoxanthine, insulin, selenium at 10 μg/mL and transferrin at 5.5 μg/mL (α-MEM+) or supplemented with 0.01, 0.1, 1, 10 and 100 µg/mL OECA at 38.5°C with 5% CO₂ in air for 6 days. Every two days, 60% of the culture medium was replaced. At the end, the tissues were fixed in 10% paraformaldehyde and processed in classical histology for staining with Hematoxylin & Eosin, Picrosirius Red and Alcian Blue, and then subjected to histological analysis for morphological assessment, follicular development, analysis of the stroma and ECM. The results indicate that the tissues cultured with 1.0, 10.0 and 100.0 μg/mL of OECA showed a higher percentage of normal follicles compared to the other treatments with 0.01 and 0.1 OECA and α-MEM+. Treatment with 100 μg/mL improved the amount of stromal cells after culture when compared to the α-MEM+ cultured control. All OECA treatments maintained collagen fiber levels when compared to the uncultured control; however, there was an increase in collagen fibers in the OECA treatments when compared to the α-MEM+ cultured control. A reduction in collagen density was observed around primary follicles compared to primordial follicles, regardless of the OECA treatment. Cultivation with OECA or α-MEM+ did not interfere with glycosaminoglycan levels when compared to the uncultivated control and cultured. It is concluded that 100.0 μg/mL of OECA favors the maintenance of follicular morphology, the integrity of stromal cells and preserves the collagen fibers and glycosaminoglycans in the extracellular matrix.