Influência da acetil-l-carnitina no desenvolvimento de folículos ovarianos pré-antrais bovinos cultivados in vitro

Abstract

The in vitro culture of ovarian follicles aims to provide growth and development of preantral follicles. One of the main obstacles to the in vitro cultivation of ovarian follicles is the excess formation of reactive oxygen species (ROS), which cause oxidative stress (OS). There is therefore a need to supplement in vitro culture media with antioxidant substances. To this end, acetyl-L-carnitine has antioxidant and anti-apoptotic potential and can help reduce EROS and DNA damage during in vitro cultivation. Therefore, the aim of this study was to evaluate the effects of acetyl-L-carnitine during the in vitro culture of preantral follicles embedded in bovine ovarian tissue. The experimental study was approved by the Ethics Committee for the Use of Animals - CEUA of the Federal University of Ceará Sobral Campus under protocol N◦ 03/22. To this end, bovine ovaries from 16 cows were subjected to fragmentation of the ovarian cortex. Part of the fragments were fixed in 4% paraformaldehyde (uncultured control) or cultured in control medium (α-MEM⁺) alone or supplemented with 10, 50 or 100 µM acetyl-L-carnitine at 38.5°C, 5% CO2, for 6 days. At the end of culture, cultured and uncultured tissues were sent for classical histology to assess morphology, follicle activation and growth, tromal cell density, and collagen content in the extracellular matrix. In addition, mRNA levels for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), peroxiredoxin 6 (PRDX6) and nuclear factor erythroid 2 (NRF2) were assessed by real-time PCR. The data obtained was analyzed using GraphPad Prisma (9.0). The percentage of normal, primordial and developing follicles was analyzed using the Chi-square test. The other evaluations were carried out using the Kruskal-Wallis test. Differences were considered significant when P<0.05. The results showed that tissues cultured in the presence of 100 µM acetyl-L-carnitine had significantly higher levels of morphologically normal follicles than those cultured in α- MEM+ (P<0.05). In addition, tissues cultured in 100 µM acetyl-L-carnitine showed a higher density of stromal cells when compared to the non-cultured control, while for activation, collagen levels and CRP, there were no significant differences (P<0.05). In conclusion, 100 µM acetyl-L-carnitine improves follicular morphology and maintains the density of ovarian stromal cells after 6 days of culture.