Influence of adding zeolite loaded with different charges to semen extender on sperm quality in rabbits after...

The aim of the present study was to investigate the effect of supplementing rabbit semen extender with zeolite loaded with different charges (Z⁺ or Z^-, Z^±) on sperm cryopreservation. Semen was collected from six healthy, fertile New Zealand rabbit bucks using an artificial vagina. The collected ejaculates were pooled and diluted with a tris-yolk fructose (TYF) extender supplemented with Z^± (+16, +12, +8, -16, -12, and -8) at a concentration of 1% for a final sperm concentration of 25 × 10⁶ sperm cells/mL. The diluted semen samples were then cryopreserved in 0.25 mL straws and stored in liquid nitrogen for 1 month. To evaluate sperm quality, we examined sperm progressive motility, vitality, morphological abnormalities, and plasma membrane integrity. In addition, apoptotic rates were determined using flow cytometry and by examining sperm ultrastructure under a transmission electron microscope (TEM). Moreover, total antioxidant capacity and markers of lipid peroxidation were measured in the extender after thawing. Addition of Z^± had a positive effect on progressive motility, vitality, and membrane integrity after an equilibration period and post-thawing as compared with the controls (P < 0.05). Z^± supplementation, particularly with a strong negative charge, also decreased the percentages of apoptotic and necrotic sperm cells compared to controls (P < 0.05), as shown both by flow cytometry and TEM. This was not associated with any marked effects on the oxidative biomarkers in the extender. In conclusion, addition of Z^± to semen extender improved post-thawing sperm quality by improving sperm characteristics, decreasing apoptosis, and minimizing sperm damage during cryopreservation.