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 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. CLC and CLC + Iodixanol significantly preserved total motility (%), acrosomal integrity (%), viability (%), and plasma membrane functionality (%) after thawing (p 0.05). Although both additives showed potential in supporting in vitro embryonic development (p 0.05). 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.
ARICI et al. (2026) studied this question.