Among the Assisted Reproductive Technologies (ART) in farm animals, in vitro maturation (IVM) of oocytes is the first and most crucial phase in the process of in vitro embryo production (IVP). During this phase, oocytes undergo nuclear and cytoplasmic maturation and acquire the potential to support further embryonic development [1]. The loss of developmental competence in in vitro-derived bovine embryos compared to their in vivo counterparts is attributed to the exposure of oocytes to various cellular stressors during the IVM process [2]. Several factors, including the intrinsic quality of the oocytes [3] and the culture conditions [4] affect the success of IVM and thus embryos development following IVF.

In bovine, IVM is commonly performed in incubators adjusted at 5% CO2 and atmospheric oxygen (O2) levels, significantly higher than the ovarian intrafollicular O2 content [5]. Additionally, IVM media is often used to transport oocytes from ovary collection or ovum pickup (OPU) stations to laboratories exposing the oocytes to higher O2 content in IVM, which triggers excessive ROS generation, leading to intracellular damage of the oocyte and surrounding cells, compromising its developmental competence, and causes embryonic loss [6].

Under normal physiological conditions, producing an optimal amount of ROS is necessary for various cellular functions. However, the production of excessive amounts of ROS compromise cellular function and viability [7], leading to oxidative stress. The level of cellular damage from ROS depends on maintaining the balance between the production of the free radicals and the cells’ ability to scavenge these free radicals and achieve cellular redox homeostasis. One strategy to reduce the adverse effects of excessive ROS during oocyte maturation is to exogenously supplement the IVM media with a variety of antioxidants proven to have a beneficial effect [8].

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), is a polyphenolic compound found in various plant species, and is one of the most widely used antioxidants. Resveratrol improves mitochondrial functions in a SIRT1-dependent manner and maintains cellular homeostasis [9]. Supplementation of resveratrol into oocyte maturation media is reported to reduce the accumulation of ROS and improve the maturation, blastocyst rate, and increase total cell number of blastocysts [10]. Similarly, resveratrol is reported to enhance the post-warming development of frozen embryos by improving mitochondrial clearance and reducing the mitochondrial copy number in blastomeres without affecting the ATP levels [11]. This makes resveratrol an attractive antioxidant choice due to its wide spectrum of positive effects in ART procedures in bovine.

Mitochondrial health is critical for several cellular functions. They serve as sensors of cellular stress, mainly oxidative stress [12]. Mitochondrial damage in the oocyte is reported to cause a carryover negative effect on embryos. Interestingly, embryos derived from oocytes that lack cellular machinery for the biogenesis and removal of damaged mitochondria, through mitophagy showed to have decreased mitochondrial potential and lower ATP generating metabolites [13]. Bovine embryos derived from fatty acid-free exposed oocytes showed higher ROS generation, lower developmental rates and carryover alterations in mitochondrial function [14], even though the embryo culture conditions were fatty acid-free [15]. This signifies protecting oocytes from ROS-induced stress during the maturation phase is essential for better embryonic developmental outcomes. Mitoquinone (MitoQ) is a commonly used mitochondria-targeted antioxidant with a higher affinity for accumulation in the mitochondrial matrix [16]. It was shown that bovine oocyte exposure to palmitic acid resulted in oxidative stress and hampered embryo development. Interestingly, supplementation of MitoQ prevented mitochondrial and oxidative stress damage, improving the blastocyst rate [17]. The addition of MitoQ to the mouse IVM media showed a positive effect in enhancing the fertilization and blastocyst rate. In addition, GSH content and mitochondrial membrane potential were increased, and the intracellular ROS concentration decreased in response to MitoQ supplementation [18].

The endoplasmic reticulum (ER) is an important organelle involved in regulating protein folding [19]. Several stressors, including ROS, disrupt the ER microenvironment and trigger ER stress, causing misfolding and unfolding of proteins and their accumulation in the ER lumen [20]. Similarly, ER stress can also cause excessive production of mitochondrial ROS, leading to the activation of apoptotic pathways [21]. Tauroursodeoxycholic acid (TUDCA) is an ER stress inhibitor that has been used to alleviate ROS-induced stress during in vitro maturation and embryo development [22,23]. TUDCA supplementation in the embryo culture media improved the developmental competence of cloned bovine embryos [24] and relieved the stress of embryos cultured under higher O2 tension [25].

To date, several antioxidant products have been reported to have beneficial effects during oocyte maturation and embryonic development (reviewed in Ref. [26]). However, differences in biochemical characteristics and modes of action of the individual antioxidants in the cells make it almost impossible to pick a single antioxidant that can work in all culture conditions and laboratory settings. Thus, it is imperative to look deeper into the variability in the mode of action and the subcellular sources of ROS.

Here, we aimed to develop an organelle-specific cocktail of antioxidants capable of acting at the different cellular sources of ROS, previously mentioned. Selection of the individual antioxidants as part of the cocktail considered the organelle-specific mode of action, which are relevant to oocyte maturation. Each component of the cocktail has previously been reported to support IVP comes when supplemented individually. The antioxidant cocktail we developed contains 0.1 μM of MitoQ, 100 μM TUDCA, 0.5 μM Resveratrol, and was named MTR-AO. We have supplemented the MTR-AO in oocyte maturation media. Following the in vitro fertilization, in vitro embryo development and hatching, production of reduced GSH, and expression of key stress-related genes were evaluated. In addition, the impact of the MTR-AO supplementation on IVM for oocytes derived from OPU, their embryo production, and subsequent conception rate were evaluated.