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

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.

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.

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.