Cryopreservation is pivotal for conserving genetic diversity. This study aimed to optimize cryopreservation techniques for Dandarawi rooster sperm through two experiments. Experiment I explored the optimal levels of two cryoprotectants, with or without sucrose, in two trials. Semen from 20 Dandarawi males was pooled, diluted, and divided into five aliquots. In trial 1, one aliquot remained fresh (control), while others were frozen in straws using glycerol (GLY) at concentrations of 8% and 11%, with or without 5% sucrose. In trial 2, one aliquot served as a control, while the others were frozen in pellets using a cryo-diluent containing 5% or 7% dimethylacetamide (DMA), with or without sucrose. Danadarawi hens (n=10 per group) were inseminated with fresh (control) or frozen-thawed sperm, with six repetitions of semen collection and insemination. In Experiment II, pooled semen was similarly divided. Aliquot 1 served as a control, while aliquots 2 and 3 were frozen in pellets with 5% DMA cryo-diluent, with and without 5% sucrose, respectively. Aliquots 4 and 5 were frozen in straws with 8% GLY, with and without 5% sucrose. Transmission electron microscopy (TEM) evaluated spermatozoa for cryodamage. GLY level did not significantly affect motility, fertility, or hatchability, but sucrose notably improved hatchability with 8% and 11% GLY. DMA did not significantly impact motility, although 7% DMA demonstrated the highest fertility. Sucrose enhanced hatchability with 5% DMA but not with 7% DMA. TEM analysis revealed higher levels of acrosomal damage in spermatozoa frozen with GLY compared to DMA, with sucrose consistently mitigating acrosomal injuries. Moderate damage was observed in the tail, sparing the nucleus and midpiece. In conclusion, the choice of freezing method and the inclusion of sucrose significantly influenced sperm freezing outcomes, demonstrating their critical roles in preserving sperm quality. Sucrose was particularly effective across all treatments, notably in protecting the acrosome region.