Anti-apoptotic effects of minocycline on ram epididymal spermatozoa exposed to oxidative stress

Cryopreservation causes major damage to sperm cells and thereby, increased apoptosis has been documented as physiologically programmed cells death. The goal of current study was to evaluate the anti-apoptotic effects of minocycline compared to sericin added to diluents on frozen-thawed spermatozoa. Epididymal spermatozoa isolated from 50 pairs testes with motility >80% and total morphological abnormalities <10% were pooled, divided into 9 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing minocycline (5 and 10 mg) and tert-butyl hydro peroxide (10 and 20 μm) and sericin (0.5 gr). Total sperm motility and progressive motility were evaluated by Computed Assisted System Analysis (CASA). Acrosome and plasma integrity, viability, hypo osmotic swelling test, malondialdehyde concentration, DNA fragmentation test, mitochondrial activity, apoptotic features, caspase activity and level of H2O2 and O2 were assessed as completed characteristics of frozen-thawed spermatozoa. The results indicate that 10 mg minocycline added to extender resulted in significant (P < 0.05) enhancement of total motility, progressive motility and viability of post-thawing spermatozoa. The results of plasma membrane demonstrate significantly (P < 0.05) higher value of both minocycline concentrations with 10 μm tert-butyl hydro peroxide. In regard to caspase activity, diluents supplemented with 5 and 10 mg minocycline improved significantly (P < 0.05) the rate of viable spermatozoa. The results of malondialdehyde concentration show diluents supplemented with 10 mg minocycline and 0.5 gr sericin were significantly (P < 0.05) lower than other extenders. The ratio of sperm chromatin dispersion stained spermatozoa illustrated the higher rate in extenders containing 10 mg minocycline plus 10 μm tert-butyl hydro peroxide, 5 mg minocycline plus 10 μm tert-butyl hydro peroxide and 5 and 10 mg minocycline were significantly (P < 0.05) than other treatments. Moreover, the results of mitochondrial activity show significantly (P < 0.05) an improvement by supplementation of 5 and 10 mg minocycline. Regarding the apoptosis occurrence and intracellular ROS, the results demonstrate significantly (P < 0.05) higher live post thawed spermatozoa and lowest value for diluents containing 10 and 5 mg, respectively. In conclusion, the results indicate that addition of 10 mg minocycline seems to reduce the apoptotic marker during cryopreservation of epididymal ram spermatozoa.