In recent years, ovarian tissue cryopreservation has become an excellent tool for preserving the fertility of high genetic value herds, as well as endangered wildlife species [1,2]. In addition, it is considered the only option to preserve the reproductive capacity of females who die unexpectedly or of young females who do not respond to the hormonal treatments required in assisted reproduction techniques [3]. Generally, the methods applied for this technique have been slow freezing or vitrification [4]. The latter has stood out for being a simple method, not requiring specific or sophisticated equipment that demands energy or integrated technology, making the process less costly, less complex, and more feasible for field application [5,6]. Promising studies have already been reported in different species (sheep [7]; goat [8]; and cattle [9]), including the birth of healthy individuals [10] after ovarian tissue vitrification and transplantation.

However, despite the results, transplanted ovarian grafts have a limited lifespan due to ischemia [11] with low cost-effectiveness, especially in production animals such as sheep. Therefore, the in vitro culture of ovarian tissue post-warming is a promising alternative as it allows the reconstitution of the ovarian microenvironment, providing conditions for follicular growth [12,13] and obtaining fertilizable oocytes. Previous studies have reported the birth of healthy mice after in vitro culture of fresh [14] or vitrified [15,16] ovarian tissue. However, in livestock, despite continuous efforts to culture ovarian cortical tissue, the development of applicable solid protocols remains a major challenge [17].

Unlike embryos and oocytes, ovarian tissue culture is very complex because, in addition to the different stages of follicular development, it is necessary to consider the components and various types of cells that constitute the extracellular matrix [18], responding to very different nutritional needs and cellular metabolism [19]. These characteristics combined with in vitro conditions can result in increased production of Reactive Oxygen Species (ROS), impairing follicular development and oocyte quality [20]. This effect may be more intense in cryopreserved samples, which are exposed to mechanical and chemical stresses [21]. Therefore, controlling the production of ROS during ovarian culture is one of the crucial points for in vitro follicular development.

Supplementing the culture medium with exogenous antioxidants minimizes the excessive production of ROS and maintains the homeostatic balance during in vitro culture [22]. Alpha lipoic acid (ALA), a disulfide molecule that plays an important role in regulating mitochondrial function, is an antioxidant that can increase the rate of viable follicles during in vitro culture. In addition, its reduced form (dihydrolipoic acid) acts by eliminating free radicals and indirectly recycling other intracellular antioxidants such as glutathione, catalase, vitamins C, and E; protecting membranes against lipid peroxidation [23,24]. ALA has been added to the in vitro culture medium of fresh ovarian tissue (bovine [25]; equine [26]), resulting in a reduction in ROS levels, preservation of morphological integrity, and follicular development. Recently, we have successfully used ALA in the vitrification solution of sheep ovarian tissue [5]. However, we have no report of the action of this antioxidant during culture after warming the ovarian tissue.

Therefore, this study aimed to evaluate the effects of adding ALA to the in vitro culture medium of fresh or vitrified/warming ovarian tissue. For a broad and more assertive analysis, morphology and follicular development, stromal density, DNA fragmentation, cellular senescence, fibrosis, oxidative stress, antioxidant capacity, and steroidogenesis were evaluated.