This research aimed to compare the impact of various semen extenders and freezing techniques on the post-thaw quality of sperm following cryopreservation, with the goal of identifying an affordable and effective approach for preserving Hu ram semen suitable for use on farms. Semen was collected from five Hu rams. In Experiment I, samples were diluted using eight distinct extenders (formulas A–H). After dilution and gradual cooling, 0.25 mL straws were filled and frozen by exposure to liquid nitrogen vapors. In Experiment II, diluent C was selected as the base formula, and semen was cryopreserved through liquid nitrogen fumigation and two different program-controlled freezing systems. The frozen semen was analyzed for motility indicators (total motility (TM), progressive motility (PM)), sperm kinetics (straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head movement (ALH), wobble coefficient (WOB), and mean angular displacement (MAD)), levels of reactive oxygen species (ROS), and membrane and acrosome integrity.
In Experiment I, diluent C demonstrated superior TM, PM, and structural integrity of both membrane and acrosome and lower ROS levels (p < 0.05) compared with the other extenders except A. It also exhibited greater (p < 0.05) values for VCL, VAP, ALH, WOB, and MAD compared with B, D, E, and F. In Experiment II, there were no significant differences (p > 0.05) among the three freezing systems for TM and the biokinetic indicators. Nevertheless, liquid nitrogen fumigation achieved higher (p < 0.05) PM, membrane and acrosome, integrity, and reduced ROS levels compared with the programmed systems. Overall, semen diluted with formula C produced higher-quality sperm after thawing. The liquid nitrogen vapor method yielded better cryopreservation outcomes than the program-controlled approaches when diluent C was applied.
