Cryopreservation of Aseel rooster sperm using different glycerol–iodixanol combinations and evaluation of post-thaw quality

BACKGROUND: Cryopreservation of avian semen is a critical tool for genetic conservation and poultry breeding programs. However, the use of glycerol, the most common penetrating cryoprotectant, is associated with dose-dependent impairments in sperm function and potential contraceptive effects in the female reproductive tract. In this study, we investigated the potential of iodixanol, a non-penetrating and biocompatible medium, to improve post-thaw sperm quality when used alone or in combination with reduced glycerol concentrations. Semen samples from ten Aseel roosters were pooled and cryopreserved (n = 10) using Beltsville Poultry Semen Extender supplemented with varying concentrations of glycerol (2.5% or 5%) and iodixanol (0%, 2.5%, or 5%) in different combinations. Post-thaw sperm quality was assessed using computer-assisted sperm analysis (CASA), eosin-nigrosin staining, hypoosmotic swelling test (HOST), and flow cytometry to evaluate mitochondrial activity, plasma membrane integrity, acrosome damage, apoptosis, and chromatin structure. RESULTS: Extenders containing 5% glycerol yielded the highest total and progressive motility and improved plasma membrane integrity. In low-glycerol groups, iodixanol supplementation significantly improved total motility, while membrane integrity and mitochondrial activity showed numerical improvement, with the 2.5% glycerol + 2.5% iodixanol group demonstrating the most favorable overall outcomes. Flow cytometric analysis further supported these findings by showing improved mitochondrial membrane potential and plasma membrane integrity, indicative of reduced cryodamage. CONCLUSIONS: Iodixanol enhances the cryoprotective efficacy of extenders, particularly under low-glycerol conditions, by improving motility, membrane integrity, and mitochondrial activity. The combined use of glycerol and iodixanol may provide a more effective cryopreservation strategy for Aseel rooster sperm, thereby supporting genetic conservation programs and improving artificial insemination outcomes in poultry breeding.