Bovine embryo transfer has been practiced for several decades, and according to the International Embryo Technology Society report [1], in vivo embryo transfers surpassed in vitro transfers until 2016. However, with the commercialization of the OPU-IVF system, in vitro embryo transfers have become increasingly common. Compared to in vivo embryos, in vitro embryos exhibit distinct differences in metabolism [2], gene expression [3], and mitochondrial number [4], making them more sensitive to freezing and, therefore, resulting in lower conception rates [5,6]. As the prevalence of in vitro embryo transfers continues to grow, it is essential to improve the culture and freezing medium to enhance the success of these procedures.

Recent studies have reported preimplantation genetic testing of bovine embryos [7], highlighting the need for effective freezing technologies to support such advancements. Furthermore, embryo cryopreservation is a pivotal strategy for the introduction of novel genetic resources into the gene pool of a particular region or country. During fresh embryo transfers, estrus synchronization is used to align the uterine environment of the recipient with the embryo; however, frozen embryos can be successfully transferred even during natural estrus. Embryo freezing methods can be divided into rapid freezing and slow freezing. Rapid freezing involves the use of high-concentration cryoprotectants to freeze the embryo in a vitrified state, resulting in higher survival and conception rates when compared with slow freezing [8]. However, due to the high concentration of cryoprotectants, they must be diluted during thawing, and this process requires special equipment and technology in the field. Slow freezing offers the advantage of enabling embryo transfer directly in the field while requiring lower concentrations of cryoprotectants compared to vitrification [8]. On bovine farms, embryos are typically thawed and then transplanted on-site, making slow freezing embryos the preferred choice. Despite their effectiveness, cryoprotectants used in both freezing methods are permeable and exhibit dose-dependent toxicity to cells [9], potentially exerting detrimental effects when exposure is prolonged. Additionally, the freezing process exposes embryos to oxidative stress. Compared to fresh embryos, frozen embryos have been reported to have higher levels of reactive oxygen species (ROS) [10]. Increased ROS levels in frozen-thawed embryos have been linked to increased apoptosis [10], which may compromise embryo viability. Recently, it has been demonstrated that the addition of antioxidants to the freezing medium enhances embryo viability [11,12]. Furthermore, extensive research has been conducted across various species to enhance the survival of embryos after freezing-thawing, intracellular ice crystal formation can further damage the cells [[13], [14], [15]].

L-proline (Pro) is a natural amino acid with various biological functions, including cell proliferation, cell structure maintenance, and metabolism [16,17]. It is highly soluble, has a neutral pH, and a relatively high osmotic pressure [18]. Pro is non-toxic even at high concentrations, making it an ideal osmotic agent and cryoprotectant. It also displays antioxidant properties, which reduce ROS levels in mouse oocytes during in vitro maturation [19]. Pro has been used to preserve donkey [20], boar [21], and goat sperm [22]. Additionally, the preservation of mouse oocytes using vitrification solutions with the addition of Pro has been reported [23]. However, its use in slow-freezing medium for bovine embryos has not yet been explored.

Sucrose (Suc), commonly known as sugar, is a naturally occurring disaccharide comprising equal parts fructose and glucose, which are linked by glycosidic bonds. It is classified as a non-penetrating cryoprotectant that functions by dehydrating intracellular water and protecting cells from osmotic shock when exposed to the freezing medium [24,25]. In slow freezing, the combination of ethylene glycol (EG) and Suc has been controversial due to its impact on embryo survival and implantation rates in previous studies [26,27]. Furthermore, trehalose (Tre) has been documented as a noninvasive cryoprotectant. In a study conducted by Somfai et al. [28], the developmental rate of porcine oocytes was examined following supplementation with Tre and Suc in the vitrification medium. However, no significant differences were observed in the results. While a direct comparison with Pro, Suc, and Tre remains to be established, Pro is a hyperosmotic substance that also possesses non-toxic properties as a cryoprotectant. Furthermore, it is a natural amino acid with antioxidant properties, suggesting its potential to replace Tre and Suc and serve as a promising alternative cryoprotectant in the future.

To the best of our knowledge, the effect of Pro on the slow freezing of bovine embryos is unknown. In this study, we hypothesized that the addition of Pro to slow freezing medium would protect embryos from cryoprotectant toxicity and reduce ROS levels by acting as an antioxidant. Therefore, we aimed to evaluate the viability and quality of blastocysts after slow freezing-thawing using freezing medium containing Pro and EG. Additionally, we investigated the relationship between Pro and Suc in the freezing medium.