Vitrification is a highly efficient cryopreservation technique widely applied in biotechnology, particularly in the preservation of biological samples [[1], [2], [3]]. Compared to conventional slow-freezing methods, vitrification prevents ice crystal formation, thereby minimizing cellular damage. This technique is extensively used for preserving human oocytes, embryos, and various animal germplasm resources, playing a crucial role in assisted reproductive technologies (ARTs) [4]. Recent advancements in microscopy and cryoprotectant formulations have promisingly expanded the scope and efficiency of vitrification, offering robust solutions for the long-term storage of cells and tissues. These developments have had profound implications for biomedicine and bio-agriculture.

Despite its advantages, vitrification-induced cryoinjury compromises the oocyte developmental potential in mammals. For instance, the blastocyst formation rate of vitrified bovine metaphase II (MII) oocytes is nearly 10 % lower than that of fresh oocytes [5]. Several factors contribute to this decline, including ice crystal formation, cryoprotectant toxicity, osmotic stress from low temperatures, and oxidative stress due to excessive intracellular reactive oxygen species (ROS) accumulation [6,7]. Among these, oxidative stress is particularly detrimental, as vitrification triggers severe mitochondrial dysfunction and ROS overproduction in oocytes [8].

ROS generated during oocyte cryopreservation originate from both intracellular and environmental sources. Mitochondrial complexes I and III in the respiratory chain are the primary intracellular ROS producers during oxidative metabolism [9]. Additionally, enzymatic reactions involving NADPH oxidase (NOX) and nitric oxide synthase (NOS) contribute to ROS accumulation in the cytoplasm and cell membrane [7]. Endoplasmic reticulum (ER) stress also exacerbates oxidative damage, as oxidases such as NOX4 enhance ROS generation under cryogenic conditions [10]. Extracellularly, cryoprotectants such as dimethyl sulfoxide (DMSO) induce massive calcium ion (Ca2+) release from the ER into the cytoplasm, leading to increased mitochondrial Ca2+ uptake and further amplifying ROS production [[11], [12], [13]]. The cryogenic nature of vitrification protocols exacerbates these effects, promoting mitochondrial oxidative stress and compromising oocyte viability [14].

To counteract oxidative stress, mammalian cells rely on three key enzymatic antioxidants: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). SOD catalyzes the dismutation of superoxide radicals (O2−) into hydrogen peroxide, which is subsequently neutralized by CAT or GPX. Upregulation of SOD has been observed in vitrified mouse and porcine oocytes, enhancing ROS scavenging capacity [11]. Elevated CAT activity in vitrified ovine oocytes and the protective role of CAT supplementation in vitrified goat ovarian tissue further underscore the importance of enzymatic antioxidants in mitigating cryopreservation-induced oxidative damage [15,16]. Mitochondria also play a central role in ROS-mediated oocyte damage during vitrification. Cryopreservation disrupts mitochondrial function, impairing the electron transport chain and causing electron leakage, a primary source of ROS generation [17]. Dysfunctional mitochondria fail to completely reduce oxygen, leading to the formation of reactive species such as superoxide anions. Furthermore, cryopreservation may impair the mitochondrial antioxidant defense system, diminishing the cell's ability to neutralize metabolically generated ROS and exacerbating intracellular oxidative stress [18]. Excessive ROS accumulation damages critical biomolecules, adversely affecting oocyte quality and developmental potential [19]. Consequently, antioxidant supplementation during vitrification has emerged as a promising strategy to mitigate ROS-induced damage.

Glutathione (GSH), a tripeptide composed of glutamic acid, cysteine, and glycine, is a pivotal non-enzymatic antioxidant that plays a crucial role in cellular redox homeostasis. It neutralizes free radicals, regenerates other antioxidants, and detoxifies harmful compounds [20]. Within oocytes, GSH, along with its precursors cysteine (CYS) and cysteamine (CSH), constitutes a key defense system against oxidative stress [18]. Notably, GSH has been shown to mitigate spindle damage caused by oxidative stress during in vitro maturation [21]. Cysteamine, a small thiol compound that enhances GSH synthesis, has been incorporated into bovine oocyte maturation media to counteract oxidative damage by increasing intracellular GSH levels [22]. Additionally, cysteamine directly scavenges hydroxyl radicals (-OH), thereby maintaining redox balance and sustaining a high GSH/GSSG ratio in oocytes [23].

Given the critical role of GSH in redox regulation, we hypothesized that GSH supplementation improves the vitrification survival rate of ovine oocytes by mitigating oxidative stress, preserving mitochondrial function, and reducing meiotic defects. To elucidate the underlying mechanisms, we conducted a comprehensive analysis of oocyte developmental parameters, including nuclear maturation, spindle and chromosome alignment, cytoskeletal integrity, cortical granule dynamics, mitochondrial function, ROS generation, and apoptosis. Furthermore, we evaluated the developmental competence of vitrified ovine oocytes through cleavage assays and embryonic development assessments. Collectively, these investigations provide mechanistic insights into the role of GSH in enhancing oocyte survival and quality following vitrification.