Role of Antioxidants in Bovine and Buffalo Oocyte Maturation: From Redox Homeostasis to Developmental Competence

Abstract

INTRODUCTION: Bovine (Bos taurus and Bos indicus) and buffalo (Bubalus bubalis) are economically important livestock species and major contributors to milk and meat production. Assisted reproductive technologies, particularly in vitro embryo production (IVEP), provide important opportunities for the rapid multiplication of genetically superior animals and efficient dissemination of desirable genetic traits. However, the efficiency of IVEP remains limited, and oocyte developmental competence is a major determinant of subsequent fertilization, embryo development and blastocyst formation. In vitro maturation (IVM) is therefore a critical component of IVEP, involving coordinated nuclear and cytoplasmic maturation, organelle redistribution, metabolic remodelling and preparation of the oocyte cytoplasm for fertilization and early embryogenesis (Ferreira et al., 2009; Roelen, 2019). Unlike the physiological follicular environment, conventional IVM conditions expose oocytes to altered oxygen tension, temperature, pH, light and nutrient composition, which may disturb cellular redox balance. Reactive oxygen species (ROS) are physiological products of cellular metabolism and participate in intracellular signalling; however, excessive ROS generation relative to antioxidant capacity results in oxidative stress (OS). Elevated ROS can induce lipid peroxidation, protein oxidation and genomic and mitochondrial DNA damage, impair mitochondrial function and alter cellular signalling, thereby compromising oocyte quality and subsequent embryo development (Keane and Ealy, 2024). Maintenance of an appropriate redox state is consequently essential during IVM. Endogenous antioxidant systems, including glutathione (GSH) and enzymatic antioxidants provide intrinsic protection against ROS, whereas exogenous antioxidants can supplement these defenses under culture conditions. In bovine oocytes, supplementation with quercetin, cysteamine, carnitine, vitamin C or resveratrol during IVM altered intracellular ROS and/or GSH levels and was associated with improved blastocyst development compared with non-supplemented controls (Sovernigo et al., 2017). The importance of redox regulation is also evident in buffalo. El-Sanea et al. (2021) demonstrated that maturation under 5% oxygen improved maturation, cleavage and blastocyst rates compared with culture under 20% oxygen. Furthermore, supplementation with ascorbic acid or melatonin under 20% oxygen improved cumulus expansion, nuclear maturation and subsequent embryo development, indicating that antioxidant supplementation can partially counteract oxidative challenges during buffalo IVM. Thus, antioxidant-mediated modulation of redox homeostasis represents an important strategy for improving oocyte quality and developmental competence. This chapter examines oxidative stress during bovine and buffalo IVM, endogenous and exogenous antioxidant defenses, mechanisms of antioxidant action, effects on mitochondrial function and oocyte maturation, and implications for subsequent embryo development. Cellular Stress During In Vitro Maturation of Bovine and Buffalo Oocytes: In vitro maturation (IVM) exposes bovine and buffalo cumulus–oocyte complexes (COCs) to an artificial environment that differs substantially from the tightly regulated follicular microenvironment. Although IVM systems support meiotic progression, deviations in oxygen tension, temperature, pH, nutrient availability and metabolic substrates can disturb cellular homeostasis and compromise oocyte developmental competence. These disturbances generate interconnected forms of cellular stress, principally oxidative, thermal, mitochondrial, endoplasmic reticulum (ER), metabolic and lipotoxic stress. 2.1 Oxidative stress is one of the most extensively characterized challenges during IVM. Reactive oxygen species (ROS), generated predominantly through cellular metabolism and mitochondrial oxidative phosphorylation, have physiological roles in signaling; however, excessive ROS relative to antioxidant capacity disrupts redox homeostasis. Increased ROS can promote lipid peroxidation, protein oxidation, DNA damage, mitochondrial dysfunction and apoptosis, thereby impairing oocyte quality and subsequent embryo development. The relatively high oxygen concentration used in conventional culture compared with the follicular environment further contributes to oxidative imbalance (Keane and Ealy, 2024). 2.2 Thermal stress is another important determinant of oocyte competence, particularly in cattle. Exposure to elevated temperature during IVM can disturb meiotic progression, mitochondrial activity and cellular redox balance and can activate heat-shock and apoptotic responses. Importantly, heat stress during bovine oocyte maturation has been associated with altered mitochondrial bioenergetics, increased ROS and ER stress in subsequent embryos, indicating consequences extending beyond the maturation period (Roth, 2017; Held-Hoelker et al., 2025). 2.3 ER stress develops when protein-folding capacity is exceeded, resulting in accumulation of unfolded proteins and activation of the unfolded protein response. Persistent ER stress can promote oxidative imbalance and apoptosis. Attenuation of ER stress during bovine IVM has been shown to improve maturation and subsequent blastocyst development, supporting its role in determining oocyte competence (Khatun and Yamanaka, 2020). 2.4 Metabolic and lipotoxic stress are particularly relevant to bovine oocytes, which contain abundant lipid reserves. Altered glucose and fatty-acid metabolism can disrupt ATP production, mitochondrial function and redox regulation. Excess saturated fatty acids, particularly palmitic acid, can induce ROS generation, mitochondrial dysfunction and apoptosis, whereas intact cumulus cells provide metabolic and protective support to the maturing oocyte (Aardema et al., 2015; Pawlak et al., 2024). Collectively, these stressors form an interconnected network in which oxidative, mitochondrial, ER and metabolic disturbances reinforce one another. Maintenance of redox, mitochondrial, protein-folding and metabolic homeostasis is therefore fundamental to successful IVM and acquisition of developmental competence. Understanding these interconnected stress pathways provides the biological basis for antioxidant and other cytoprotective strategies aimed at improving bovine and buffalo IVEP outcomes. Strategies to Mitigate Cellular Stress During In-Vitro Maturation: Different forms of cellular stress during in-vitro maturation (IVM) can be mitigated through optimization of the culture environment and supplementation with protective molecules. Oxidative stress may be reduced by lowering oxygen tension toward physiological levels and incorporating antioxidants such as melatonin, ascorbic acid, glutathione, or cysteamine (El-Sanea et al., 2021; Keane and Ealy, 2024). Thermal stress can be minimized by maintaining a stable physiological temperature, thereby limiting mitochondrial dysfunction, oxidative damage, and apoptotic responses (Roth, 2017). Endoplasmic reticulum stress may be alleviated through appropriate culture conditions and ER-stress modulators such as tauroursodeoxycholic acid (TUDCA), which can improve oocyte maturation and reduce ROS (Khatun et al., 2020). Metabolic and lipotoxic stress can be controlled by maintaining suitable energy substrates and preserving cumulus-cell integrity, which protects oocytes against fatty-acid-induced damage (Lolicato et al., 2015). Thus, integrated regulation of oxygen, temperature, redox balance, ER function, and metabolism is essential for maintaining oocyte developmental competence. Antioxidants Used During In-Vitro Maturation and Their Effects on Oocyte Developmental Competence: Antioxidant supplementation during in-vitro maturation (IVM) has been extensively investigated as a strategy to counteract excessive reactive oxygen species (ROS) generation and preserve oocyte developmental competence. Different antioxidants act through distinct mechanisms, including direct ROS scavenging, glutathione (GSH) augmentation, mitochondrial protection and modulation of endogenous antioxidant pathways. Importantly, their effects are not restricted to nuclear maturation and may be reflected in cytoplasmic maturation, mitochondrial activity, apoptosis and subsequent embryo development (Naspinska et al., 2023; Keane and Ealy, 2024). 4.1 Cysteine and cysteamine are important thiol antioxidants because they support intracellular GSH synthesis. In heat-stressed bovine oocytes, supplementation with 1.2 mM cysteine restored GSH concentrations, reduced ROS accumulation and rescued nuclear maturation and blastocyst formation, demonstrating particular benefits under oxidative challenge (Nabenishi et al., 2012). In a comparative bovine IVM study, cysteamine increased oocyte GSH, whereas cysteamine, carnitine, vitamin C, quercetin and resveratrol improved subsequent blastocyst production compared with controls, although nuclear maturation and hatching rates were not significantly different among treatments (Sovernigo et al., 2017). 4.2 Mitoquinone (MitoQ) provides a more targeted approach by accumulating within mitochondria. In buffalo oocytes, 0.1 μM MitoQ during IVM reduced ROS, increased mitochondrial membrane potential and expression of GPX1 and SOD2, and improved cumulus expansion and nuclear maturation. MitoQ supplementation during subsequent embryo culture additionally increased cleavage and blastocyst formation and reduced apoptosis-related changes (Sharma et al., 2024). 4.3 α-Ketoglutarate (α-KG) represents a metabolic/redox-modulating antioxidant rather than a conventional radical scavenger. Recent bovine studies demonstrated that α-KG supplementation during IVM enhanced oocyte maturation and embryo developmental competence, accompanied by alterations in carbohydrate, amino-acid and lipid metabolism. In OPU-derived Bos indicus oocytes, 30 μM α-KG