Furosemide sodium enhanced AQP3 expression by inhibiting NKCC1 leading to improved vitrification freezing rates of mouse and bovine blastocysts

Furosemide sodium, a loop diuretic that inhibits the Na-K-2Cl cotransporter (NKCC1), is commonly used in clinical treatment of edema. In this study, we investigated whether furosemide sodium could improve the survival and quality of vitrified mouse and bovine blastocysts by promoting the expulsion of blastocoelic fluid, thus enhancing cryoprotectant penetration and reducing ice crystal formation. Mouse and bovine blastocysts were vitrified using equilibration solutions (ES) and vitrification solutions (VS) with or without furosemide sodium, then thawed for survival and quality assessment. Results indicated that furosemide sodium significantly improved post-thaw survival rates (P < 0.05). It also markedly reduced reactive oxygen species (ROS) levels (P < 0.05). Additionally, furosemide sodium increased glutathione (GSH) levels (P < 0.05) and enhanced mitochondrial function. The improvement in mitochondrial function was reflected in increased mitochondrial membrane potential, which are key indicators of mitochondrial health and energy status (P < 0.05). Immunofluorescence analysis revealed that furosemide sodium downregulated NKCC1 protein levels and upregulated Aquaporin 3 (AQP3) protein levels (P < 0.05), indicating that furosemide sodium modulates ion transport and water regulation. This modulation likely contributes to the closure of sodium-potassium ion channels, reducing ion influx and promoting water efflux from the blastocyst. This closure limited extracellular sodium and potassium influx, significantly reducing these ions within the blastocyst cavity (P < 0.05). Similar effects were observed in both mouse and bovine blastocysts, where furosemide sodium promoted fluid expulsion from the blastocoel, thereby enhancing vitrification efficiency. These findings suggested that furosemide sodium may be a promising agent to improve vitrification efficiency in blastocysts by increasing osmotic pressure differences across the blastocoel.