The purpose of this study was to improve the quality of frozen–thawed
Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen
before cryopreservation. Semen was collected from 4 fertile bulls, using an
artificial vagina, once weekly for 6 consecutive weeks. Semen samples were
pooled, diluted with Bullxcell® extender, and supplemented with different
concentrations of MT (0 as control, 5, 10, 20, 40, and 80  μM) before cooling,
equilibration, and freezing procedures. The frozen–thawed semen was assessed
for motility, vitality, acrosome intactness, plasma membrane integrity, DNA
integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level
and in vitro fertilizing capability. The results showed that MT at concentrations
of 10, 20, and 40  μM improved the total, progressive, and rapid motility directly
after thawing while, at the highest tested concentration (80  μM), it decreased
the progressive and rapid motility after 1, 2, and 3  h of incubation. The sperm
kinetics including STR and LIN were noticeably increased at concentrations
of 10, 20, and 40  μM directly after thawing (0  h), whereas the MT effect was
variable on the other sperm kinetics during the different incubation periods.
MitoTEMPO improved the sperm vitality at all tested concentrations, while the
acrosomal and DNA integrity were improved at 20  μM and the mitochondrial
membrane potentials was increased at 80  μM. The cleavage and blastocyst
formation rates were significantly increased by using semen treated with 20  μM
MT compared with controls. These findings suggest a potential use of MT mainly
at a concentration of 20  μM as an additive in the cryopreservation media of bull
semen to improve sperm quality.