Vitrification is an important tool to store surplus embryos in assisted reproductive technology (ART). However, vitrification increases oxidative damage and results in decreased viability. Studies have reported that L-glutathione (GSH) supplementation improves the preimplantation development of murine embryos. Glutathione constitutes the major non-protein sulphydryl compound in mammalian cells, which confers protection against oxidative damage. However, the effect of GSH supplementation on embryonic vitrification outcomes has yet to be reported. This study aims to determine whether GSH supplementation in culture media improves in vitro culture and vitrification outcomes, as observed through embryo morphology and preimplantation development. Female BALB/c mice aged 6–8 weeks were superovulated through an intraperitoneal injection of 10 IU of pregnant mare serum gonadotrophin (PMSG), followed by 10 IU of human chorionic gonadotrophin (hCG) 48 h later. The mated mice were euthanized by cervical dislocation 48 h after hCG to harvest embryos. Two-cell embryos were randomly assigned to be cultured in either Group 1 (GSH-free medium), Group 2 (GSH-free medium with vitrification), Group 3 (0.01 mM GSH-supplemented medium), or Group 4 (0.01 mM GSH-supplemented medium with vitrification). Non-vitrified (Groups 1 and 3) and vitrified (Groups 2 and 4) embryos were observed for morphological quality and preimplantation development at 24, 48, 72, and 96 h. In the non-vitrified groups, there were significant increases in the number of Grade-1 blastocysts in GSH cultures (p < 0.05). Similarly, in the vitrified groups, GSH supplementation was also seen to significantly increase blastocyst formation. Exogenous GSH supplementation resulted in a significant increase in intracellular GSH, a release of cytochrome c from mitochondria, and a parallel decrease in intracellular reactive oxygen species (ROS) levels in vitrified eight-cell embryos (p < 0.05). GSH supplementation was shown to upregulate Bcl2 expression and downregulate Bax expression in the vitrified preimplantation embryo group. The action of exogenous GSH was concomitant with an increase in the relative abundance of Gpx1 and Sod1. In conclusion, our study demonstrated the novel use and practical applicability of GSH supplementation for improving embryonic cryotolerance via a decrease in ROS levels and the inhibition of apoptotic events by improvement in oxidative status.

Vitamin E contains isomers of tocotrienols and tocopherols. Studies have shown that palm tocotrienol-rich-fraction (TRF) improves preimplantation development of murine embryos. The aim of this study is to investigate the effects of TRF and α-tocopherol supplementation on preimplantation embryonic morphology, development and mitochondrial ultrastructure. Female C57Bl/6 mice were supplemented with 60 mg/kg body weight per day TRF and α-tocopherol for seven days. The females were superovulated and mated with fertile males to obtain 2-cell stage embryos. Initial assessment of normal and abnormal morphology was carried out on 2-cell embryos. The embryos were then cultured until the blastocyst stage. At the 8-cell stage, embryos were subjected to Transmission Electron Microscopy (TEM) to observe their mitochondria. Results showed that palm TRF produced significantly higher numbers of normal 2-cell embryos compared with α-tocopherol (80.9% vs 31.4%) at p< 0.01. Alpha-tocopherol produced higher survival rate to the blastocyst stage compared with palm TRF (42.2% vs 20.6%) at p< 0.01. The TRF group showed more vacuolated mitochondria at 8-cell stage compared to the α-tocopherol group, which may have contributed to a decline in preimplantation survival rates.

The timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early have higher developmental viability compared to their late counterparts. During embryonic development, cleavage is affected by cellular metabolic processes performed by mitochondria and its synergistic interaction with endoplasmic reticulum (ER). However, in depth study on differences of mitochondria and ER ultrastructures in early- cleaving (EC) versus late- cleaving (LC) embryos is limited. This study compares mitochondria and ER ultrastructures of EC versus LC embryos using Confocal Laser Scanning Microscopy (CLSM) and Transmission Electron Microscopy (TEM). Embryos were obtained from female ICR superovulated mice, 28-30 hours post hCG. Two-cell embryos were categorized as early-cleaving (EC), while zygotes with the second polar body and two pronuclei present were categorized as late-cleaving (LC). The LC embryos were cultured in vitro until the 2- cell stage. In EC embryos, mitochondria were mostly found at the perinuclear region and closely associated with dense ER. Meanwhile, mitochondria of LC embryos were distributed uniformly within the cytoplasm. Mitochondrial fluorescence intensity was significantly higher in EC versus LC [(18.7 ± 0.4) versus (14.6 ± 0.4)] x 105 pixel, (p<0.01). Development to the blastocyst stage was also significantly higher in EC compared to LC embryos (96.7% versus 60.9%) (p<0.01). Higher viability of EC embryos is attributed to the close association of their mitochondria to ER. This contributed to better mitochondrial fission, resulting in enhanced energy generating processes and preimplantation development.

In vitro embryo culture systems often expose embryos to elevated oxidative stress levels, compromising development. Glycine (Gly) and L-Proline (L-Pro), naturally occurring amino acids with antioxidant properties, have been shown to mitigate reactive oxygen species (ROS). This study aimed to assess the individual and combined effects of Gly and L-Pro supplementation on preimplantation development in mouse embryos. Two-cell stage BALB/C mouse embryos were cultured in M16 medium supplemented with either Gly or L-Pro at concentrations of 0.2, 0.4, and 0.6 mM, and a combined group of 0.6 mM Gly + 0.6 mM L-Pro. Developmental progress to the 8-cell, morula, and blastocyst stages was recorded. Mitochondrial activity and ROS levels were quantified using MitoTracker Red and H2DCFDA assays, respectively. The combined treatment group exhibited significantly higher rates of normal morphology (94.3% 8-cell, 85.7% morula, 72.9% blastocyst) and developmental competence (92.9% blastocyst formation, 55.7% hatching) compared to controls (p < 0.01). ROS levels were lowest in the combined group (22.24 ± 1.54 pixel x10^5) and mitochondrial intensities were highest (105.1 ± 5.83 pixel x10^5), indicating improved mitochondrial activity and reduced oxidative stress. These findings suggest a complementary interaction between Gly and L-Pro in enhancing embryo quality and development by modulating oxidative stress and mitochondrial function. This novel supplementation strategy could inform future improvements in assisted reproductive technologies.