Efeito do extrato de Aloe Vera no crescimento, viabilidade e na expressão gênica de folículos secundários bovinos cultivados in vitro

Abstract

The aim of this study was to investigate the effect of different concentrations of Aloe vera extract on growth, morphology, viability, antrum formation and mRNA levels for SOD, CAT, GPX1 and PRDX6 in vitro cultured bovine secondary follicles. For this, the ovaries of multiparous cows were obtained from a local slaughterhouse. In the laboratory, the ovarian cortex was fragmented, and the secondary follicles (150 to 250µm) were isolated and cultured individually at 38.5°C, CO2 at 5%, for 18 days in a control medium consisting of TCM-199 supplemented with BSA, insulin, transferrin, selenium, FSH, penicillin and streptomycin, ascorbic acid, glutamine and hypoxanthine. The treatments consisted of adding 2.5, 5.0, 10.0 or 20.0% of the Aloe vera extract to the culture medium. Every 6 days, Follicular growth (diameter and daily growth), morphology and antrum formation was evaluated. At the end of the culture, follicular viability (fluorescence with calcein / ethidium), and the expression of transcripts for antioxidant enzymes SOD, CAT, GPX1 and PRDX6, by real-time PCR, were evaluated. Data on follicular growth and daily growth rate were submitted to the Kolmogorov-Smirnov test, followed by the Kruskal-Wallis test. To analyze survival and antrum formation, the Chi-square test was used. The Student's t-test was used to assess the levels of luminous intensity for Calceina-AM and homodimer- ethidium. The mRNA levels were analyzed by the Kruskal-Wallis test. Differences were considered significant when P <0.05. The results showed that Aloe vera maintained follicular morphology similar to the control group. Follicles cultured the presence of Aloe vera showed a progressive increase in follicular diameter until the 12 day of culture. An increase in daily growth rate was observed from day 0 to day 6 in treatment with 5.0% Aloe vera when compared to the control group. Follicles cultured with 2.5% Aloe vera had higher rate of antrum formation than those cultured in the control group. Regarding viability, follicles cultured with 2.5 and 5.0% Aloe vera showed a higher viability rate than follicles cultured in control medium. The presence of 2.5, 5.0 and 20.0% Aloe vera in the culture medium maintained the levels of mRNA for SOD, CAT and PRDX6 in follicles similar to the control group. In conclusion, Aloe vera maintains the morphology of secondary follicles cultured in vitro for 18 days, and 2.5% of Aloe vera promotes antrum formation, increases follicular viability and maintains mRNA expression for SOD and PRDX6 after cultivation.