Avaliação do efeito do timol durante a vitrificação de folículos pré-antrais inclusos em tecido ovariano bovino

Abstract

Cryopreservation is a widely used strategy for preserving fertility; however, vitrification still has limitations associated with mechanical, chemical, and oxidative stress, which can compromise tissue integrity after rewarming. In this context, antioxidant supplementation has been proposed as a promising approach to minimize cryoinjuries. The present study aimed to evaluate the dose-response effects and antioxidant potential of thymol during the vitrification of bovine ovarian tissue. Initially, a dose-response assay was conducted on bovine cumulus cells, cultured in vitro and exposed to different concentrations of thymol (2.5, 25, and 250 μg/mL), in order to evaluate cell viability and cytotoxicity using calcein-AM and ethidium homodimer-1 assays. Next, fragments of bovine ovarian cortex were fixed in 10% paraformaldehyde (non-vitrified control) or subjected to vitrification in solutions supplemented with thymol at the same concentrations. After storage in liquid nitrogen, the tissues were rewarmed in solutions with decreasing concentrations of sucrose; part of the samples was analyzed immediately after rewarming, while the rest were incubated in vitro for 24 h. Tissue integrity was assessed by follicular morphology, ovarian stromal density, and extracellular matrix components, including collagen and glycosaminoglycans. The redox state was analyzed by antioxidant enzyme activity (SOD, CAT, and GPX) and thiol and nitrite levels. The results of the cumulus cell assay showed that 25 μg/mL and 250 μg/mL of thymol increased calcein labeling compared to the control medium, which is indicative of cell viability. Vitrification of ovarian tissue promoted changes in the redox state, evidenced by thiol depletion and reduced GPX activity. Supplementation with 25 μg/mL thymol increased SOD activity in vitrified tissue, which may represent a response to oxidative stress. On the other hand, the presence of 250 μg/mL thymol preserved thiol levels and GPX activity similar to those in non-vitrified tissue. Nitrite levels were reduced after vitrification in all groups, likely reflecting metabolic suppression. In addition, thymol, at all concentrations, increased the proportion of morphologically normal follicles, preserved ovarian stromal density, and maintained extracellular matrix components after rewarming. It is concluded that the presence of 250 μg/mL thymol in the vitrification solution of bovine ovarian cortex has cytoprotective effects that contribute to maintaining redox balance after rewarming, and to preserving the structural integrity of the tissue.