The impact of supplementation with calcium and naloxone to TRIS and Lepus extenders on the quality of refrigerated rabbit semen.

Maintaining sperm quality during storage is essential for optimizing reproductive efficiency in rabbit breeding. This study investigated the effects of modulating calcium metabolism and the endogenous opioid system on rabbit sperm function. Two commercial extenders (TRIS and Lepus) were supplemented with calcium chloride (CaCl2) and naloxone (Nx), either individually or in combination. Semen was collected from 20 New Zealand White (NZW) males, pooled, diluted, and assigned to eight experimental treatments. Sperm motility, kinematic parameters, vitality, and morphology were assessed after storage, and data were analyzed by two-way ANOVA. The results showed that treatment was the main factor affecting sperm quality parameters, whereas the effect of diluent was significant only for selected variables. The results revealed significant differences among experimental groups, with naloxone-treated samples (alone or combined with CaCl2) showing the highest sperm quality. In particular, total motility (MOT) increased from approximately 56-71% in control and CaCl2 groups to about 77-85% in Nx-treated groups, while progressive motility (PMOT) improved from approximately 44-57% to 73-82%. Similarly, kinetic parameters (e.g., VAP and VCL) were markedly enhanced in Nx and Nx + CaCl2 groups (reaching ~60-69 μm/s and ~150-159 μm/s, respectively), whereas CaCl2 alone consistently reduced all motility-related parameters. Sperm vitality also improved in Nx-containing treatments (up to ~0.95-0.96), compared to lower values in CaCl2-only groups (~0.77-0.92), while the percentage of abnormalities was reduced, particularly in TRIS-based and Nx-supplemented samples (down to ~2-4%). The combined Nx + CaCl2 treatments consistently showed the best overall performance across most parameters. The synergistic effect observed in the Nx + CaCl2 groups suggests that naloxone may regulate Ca2+ influx and intracellular homeostasis, preventing detrimental effects associated with calcium overload. In conclusion, pharmacological modulation of the opioid system, particularly through low concentrations of naloxone, significantly improves rabbit semen quality and, when combined with Ca2+, further enhances sperm functional parameters. These findings provide new perspectives for optimizing artificial insemination (AI) protocols in rabbit production systems.