Background <p>This study aimed to investigate the effects of cholesterol-loaded cyclodextrin (CLC) and iodixanol, added individually or in combination to a ram semen extender, on post-thaw sperm quality, in vitro fertilization outcomes, and embryo development. Semen samples were divided into four experimental groups: Control, CLC (2&#xa0;mg CLC/120 × 10⁶ spermatozoa), Iodixanol (5%, v/v), and CLC + Iodixanol. All samples were diluted with a tris-based egg yolk extender and cryopreserved. Following thawing, sperm motility and kinetic parameters were assessed using a computer-assisted sperm analysis (CASA) system. A thermal stress test was applied, followed by re-evaluation via CASA. Sperm viability and acrosomal integrity were determined using fluorescence microscopy, and plasma membrane functional integrity was evaluated via the hypo-osmotic swelling test (HOST). Post-thaw semen samples from all groups were used for in vitro fertilization to assess cleavage and embryo development rates.</p> Results <p>CLC and CLC + Iodixanol significantly preserved total motility (%), acrosomal integrity (%), viability (%), and plasma membrane functionality (%) after thawing (<i>p</i> &lt; 0.05). Iodixanol alone did not provide significant protection, particularly regarding membrane integrity (<i>p</i> &gt; 0.05). Although both additives showed potential in supporting in vitro embryonic development (<i>p</i> &lt; 0.05), their combined use did not result in a synergistic effect (<i>p</i> &gt; 0.05).</p> Conclusion <p>Cholesterol-loaded cyclodextrin supplementation effectively enhanced post-thaw sperm quality parameters and supported in vitro embryonic development, whereas iodixanol provided limited cryoprotective benefits; however, their combination did not yield synergistic effects, indicating that further in vivo investigations are warranted to establish their practical utility in ovine reproductive biotechnology.</p>