Preserving Life: How Retinoic Acid (RA) Enhances Cell Viability and Reduces Apoptosis in Cryopreserved Blastocyst Cells of Pudong Chickens

The preservation of chicken embryonic cells is essential for protecting avian genetic resources and enhancing breeding programs. This study investigates the effects of retinoic acid (RA) on the viability, functionality, and adhesion of thawed chicken blastoderm cells (BCs) following cryopreservation. After thawing and culturing the cells for 24 h, RA treatment resulted in significantly higher cell viability and adhesion rates compared to the control group, with the 2.0 μM RA group demonstrating the best outcomes. After 48 and 72 h of culture, similar trends were observed, with the 2.0 μM RA group consistently maintaining the highest cell viability and adhesion rates. Furthermore, immunofluorescence TUNEL assays revealed that RA significantly reduced both early and late apoptosis rates, particularly at a concentration of 2.0 μM, which exhibited a strong protective effect. Flow cytometry analysis indicated that RA treatment enhanced the mitochondrial membrane potential (MMP), reflecting improved cellular health. Analysis of the apoptosis-related genes BAX, BCL-2, and Caspase-3 revealed that moderate RA concentrations promoted the expression of anti-apoptotic factors while also upregulating pro-apoptotic factors, with the 2.0 μM RA group exhibiting the highest expression levels. Cell cycle analysis showed that RA significantly influenced the distribution of BCs across different phases, with the 4.0 μM RA group exhibiting the highest proportion of cells in the G1/G0 phase, suggesting an enhanced tolerance to cryopreservation stress. Conversely, the S phase cell population was notably reduced at higher RA concentrations, indicating potential inhibition of cell proliferation. These results suggest that RA not only significantly enhances the survival rates and mitochondrial function of BCs, but also regulates the cell cycle, providing better conditions for BC cryopreservation. Overall, the addition of RA represents a valuable strategy for optimizing cryopreservation techniques in chicken embryonic cells, with implications for avian genetic resource conservation and breeding strategies.