Porcine embryo cryopreservation facilitates the utilization of valuable genetic resources and serves as a viable method for preserving embryos derived from in vitro techniques such as genetic engineering and cloning, thus supporting advancements in biotechnology and biomedicine [1]. Currently, vitrification is the most common and effective approach for cryopreserving porcine embryos at various developmental stages [2,3]. Zygotes represent the earliest totipotent stage of embryonic development, retaining the full genetic potential to differentiate into all cell lineages and form a complete organism [4]. Unlike later-stage embryos (e.g., those in the 4-cell or 8-cell stage), whose genomic activation and cell specialization have been initiated, zygotes exhibit relatively homogeneous cellular components and simple metabolic networks [[5], [6], [7], [8]]. This structural and functional simplicity makes parthenogenetically activated (PA) 1-cell embryos an ideal model for investigating the fundamental mechanisms of cryoinjury and cryoprotection during vitrification, as their response to vitrification/warming stress is less complicated by stage-specific cellular complexity. However, research regarding the cryopreservation of early-stage porcine embryos has received limited attention. It has been reported that the vitrification of in vivo-derived porcine zygotes results in more than 40% blastocyst development without any pretreatment [9]. Therefore, reducing oxidative damage during the vitrification of PA 1-cell embryos can significantly increase both the rate of blastocyst formation and its quality. Numerous studies have demonstrated that cryopreservation causes an increase in oxidative stress through the overproduction of ROS in oocytes and embryos, which may be responsible for their impaired quality and development [10]. Excessive amounts of ROS eventually result in changes at the physiological, biochemical and molecular levels, including DNA fragmentation, spindle defects, metabolic abnormalities, organelle dysfunction, and apoptosis [11,12]. Increased ROS production is also a key factor contributing to reduced oocyte viability postvitrification [13]. Additionally, various antioxidants effectively inhibit oxidative stress in oocytes and embryos during cryopreservation or subsequent in vitro culture. Commonly used antioxidants such as melatonin [14,15], resveratrol [16,17], L-Carnitine [18,19], and glutathione [20,21] have been shown to enhance oocyte and embryo potential after cryopreservation.

Glycine is an antioxidant with anti-inflammatory, immunomodulatory and cytoprotective properties [22,23]. It prevents oocyte and embryo developmental disorders induced by harmful factors (including oxidative stress and osmotic damage) at the cellular, organ, and organismal levels and plays an essential role in the reproductive system. Studies have shown that supplementation of vitrification solutions with Gly improves mitochondrial function in oocytes, reduces apoptosis, and enhances subsequent developmental potential [24,25]. For example, fully grown germinal vesicle (GV) oocytes in mice accumulate Gly via the GLYT1 transporter to regulate cell volume [26]. Previous research has also indicated that Gly supplementation alleviates osmotic stress induced by vitrification/warming, improving the development of vitrified mouse oocytes [25] by reducing ROS levels, enhancing mitochondrial function, and decreasing apoptosis, thereby increasing the in vitro maturation rate of porcine oocytes and the embryo development rate [27,28]. Despite these beneficial effects on oocyte maturation and embryo development, whether glycine can protect vitrified porcine PA 1-cell embryos through its antioxidant capacity remains unclear. On the basis of the above issues, the aim of this study was to understand how glycine administration improves the embryonic developmental potential of vitrified porcine 1-cell embryos and to confirm the cytoprotective effect of glycine on porcine PA 1-cell embryos during the vitrification and warming process.