Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol.

Simple Summary: In the rabbit species, the use of frozen semen could play a twofold role; on the one hand, it could bring many benefits to commercial breeding operations that rely on artificial insemination, and on the other hand, it would enable the conservation of biodiversity through the establishment of semen cryobanks. One of the less explored aspects of cryopreserving rabbit semen involves establishing the ideal sperm concentration in straws. Achieving this goal is crucial for minimizing the variability in outcomes and accurately determining the number of sperm delivered during artificial insemination procedures. This study sought to determine the ideal sperm concentration in straws for cryopreserving rabbit semen, with the broader goal of improving and standardizing the freezing protocol developed by our research group over the past decade. Our study provided a comprehensive analysis of how different sperm concentrations within straws (ranging from 15 to 75 million sperm) influenced the critical stages of the cryopreservation process, starting from initial dilution with the extender, through to cooling and equilibration, and culminating in the thawing phase. This investigation shed light on the role of the sperm concentration when determining the overall success of rabbit semen cryopreservation. This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 10⁶ per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 10⁶ exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 10⁶ (49.9% and 19.7%) and 35 × 10⁶ (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration. [ABSTRACT FROM AUTHOR]