Fertility and flow cytometry evaluations of ram frozen semen in plant-based extender supplemented with Mito-TEMPO.

Semen cryopreservation is an effective strategy for distributing spermatozoa for artificial insemination, but this process reduces the fertility potential of post-thawed spermatozoa. The current study was conducted to assess effects of the novel mitochondria-targeted antioxidant "Mito-TEMPO" on ram sperm quality and fertility potential during the freeze-thawing process in a plant-based extender. Semen samples were diluted in extenders supplemented with 0, 0.5, 5, 50 and 500 µM Mito-TEMPO and then frozen using a standard protocol. Motility, abnormal morphology, acrosome integrity, membrane integrity, mitochondria membrane potential, viability, apoptotic-like changes, lipid peroxidation, DNA fragmentation, H2O2 concentration and fertility potential were assessed after thawing. Results indicated that with the 5 and 50 μM Mito-TEMPO there was a greater (P ≤ 0.05) percentage of sperm total motility, progressive motility, acrosome integrity and viability as well as less (P ≤ 0.05) lipid peroxidation and late apoptotic-like changes. Membrane integrity and mitochondria membrane potential were greater (P ≤ 0.05) with the 50 μM Mito-TEMPO extender supplementation. Furthermore, with 0.5, 5 and 50 μM Mito-TEMPO supplementations there was a greater (P ≤ 0.05) average path velocity and lesser (P ≤ 0.05) percentages of spermatozoa with DNA fragmentation and relatively greater H2O2 concentration. Results from the fertility experiment indicated the 5 and 50 μM Mito-TEMPO treatments resulted in greater pregnancy, parturition and lambing rates. It, therefore, is concluded that Mito-TEMPO may enhance quality and fertility potential of post-thawed semen of rams.