Naloxon’s effects on post-thawed buffalo sperm motility, viability and capacitation.

It is generally accepted that a substantial number (about 50%) of sperm cells are damaged during cryopreservation and several studies are concentrated on research of better standards of semen cryopreservation to optimize viability and, so, the usability for artificial reproduction. Poor studies are published on possibilities to improve sperm viability after thawing. In literature, it is evidenced that the administration of opioid antagonists to raw or frozen semen significantly improves the capacity of sperm cells to acquire the conditions essential to fertilize oocytes. The aim of the present study was to evaluated the effects of naloxone (Nx), an opioid antagonist, added post-thawing, on buffalo sperm cell quality analizing as the motility by CASA system, as the viability and capacitation by Chlortetracycline (CTC)/Hoechst 33258 coloration. Straws of buffalo semen were thawed for 30 sec in a 37°C water-bath; the semen recovered was washed with a modified Tyrode medium. For the functional test, sperm cells were incubated at the concentration of 25 millions/mL at 38°C in absence (control condition) and in presence of different Nx concentrations (10-3, 10-6 and 10-8 M) or heparin (10 IU/mL) as known capacitation inducer. Viability and capacitation were detected by CTC/Hoechst 33258 staining at 0, 1 and 3 hours incubation time points. Fluorescence microscope analysis clearly allowed differentiation of dead/live and capacitated/non capacitated cells. Statistical analysis of motility data by CASA showed that only the average path velocity (VAP) at 1h was modified negatively by the concentration of 10-3 Nx (p<0.05) in respect to control condition, whereas all other parameters were not modified. Statistical analysis of viability and capacitation data revealed that Nx at the concentrations of 10-3 and 10-6 M significantly (p<0.05; 0.001 respectively) improved sperm cell viability after 1h; whereas the Nx 10-6 M on capacitation resulted efficacious after 3h of incubation in respect to control condition. Moreover 10-6M Nx at 1 and 3h statistically increased (p<0.001) the rate of viable cells compared to heparin determining, at the same time, a comparable rate of capacitated. These results indicate that Nx few or never influences buffalo sperm cell motility (only VAP at 10-3M at 1h) but at 10-6M could be considered a better capacitating agent in IVF protocols than heparin.