Effect of mitochondria‐targeted antioxidant on the regulation of the mitochondrial function of sperm during cryopreservation

Abstract

Sperm mitochondrion is one of the major susceptible organelles that get damaged during cryopreservation. The study aimed to minimize mitochondrial dysfunction and oxidative stress during sperm cryopreservation using mitochondria‐specific antioxidants. For this, semen was collected from five buffalo bulls (3 ejaculates/bull). The ejaculates were diluted in an low‐density lipoprotein‐based extender and divided into four equal aliquots. Mitochondria‐targeted antioxidant (MitoQ) was added at a final concentration of 0 (control), 0.02, 0.2 and 2 μM separately in each aliquotes and cryopreserved. The addition of MitoQ at a concentration of 0.02 μM improved post‐thaw sperm motility, plasma membrane integrity and able to sustain sperm motility for a longer time. To investigate MitoQ's effects on mitochondrial function, we measured mitochondrial membrane potential (MMP) using JC‐1 dye, superoxide production using Mitosox assay, and lipid peroxidation by TBARS assay. The supplementation of 0.02 μM MitoQ in the extender prevented the significant reduction of MMP and reduced superoxide production resulting in lower lipid peroxidation of sperm plasma membrane after cryopreservation. Further, we found that a higher concentration of MitoQ decreases MMP and increases mitochondrial superoxide production. In conclusion, MitoQ @ 0.02 μM can alleviate oxidative stress by regulating mitochondrial functionality in spermatozoa during cryopreservation.

Protective effects of different doses of MitoQ separately and combined with trehalose on sperm function and antioxidative status of cryopreserved Markhoz goat semen

“…In our study, the extenders with 200 nM of MitoQ presented better post-thawed sperm quality characteristics such as viability, acrosome and membrane integrity, MMP and DNA fragmentation, which is in agreement with previous studies [6,13,25,38,41]. This preserving sperm quality can be related to the ability of MitoQ to directly lowed ROS levels in mitochondria.…”

“…The obtained results agree with the previous study in buffalo cryopreserved semen [38], which indicated 200 nM MitoQ increased live sperms and MMP, and decreased apoptotic and necrotic sperms. Similar studies demonstrated that the MitoQ supplemented with semen freezing extender had greater live sperms with active mitochondria in catfish [13], human [21], and buffalo [6]. On the contrary, a recent report by Câmara et al [9] showed that 0.2 to 20 nM MitoQ did not affect sperm quality and mitochondrial performance after bull semen cryopreservation, which might be due to the use of low concentrations of MitoQ in this study.…”

“…Following sperm cryopreservation, the superoxide anion as the principal mitochondrial ROS increases, which promotes lipid peroxidation and ultimately impairs sperm function [37]. MitoQ has been able to diminish superoxide anions in frozen-thawed sperm, which could be explained by the enhanced mitochondrial antioxidant enzymes, especially SOD [6]. SOD and GPx enzymes act as the primary intracellular defenses against stress oxidative by detoxifying ROS.…”

“…Hence many studies have evaluated the cryoprotective effect of various exogenous antioxidants in semen extenders with target normalizing ROS levels through different oxidative pathways [5]. Exogenous antioxidants, including metabolites and ions less than 5 kDa size, are permitted to enter the external mitochondrial membrane, while none can penetrate the inner mitochondrial membrane [6,35]. Several mitochondria-targeted antioxidants have been designed to achieve direct antioxidant delivery in the inner mitochondrial membrane, which can provide higher concentrations at the ROS production site [33].…”

“…MitoQ could cross the rat blood-testis barrier and protect against rodent testicular injury by promoting the expression of mitochondrial dynamics proteins [44], activating antioxidant enzymes, and inhibiting apoptosis indicators [19]. The addition of MitoQ at various nanomolar concentrations in extender lowered the production of ROS and lipid peroxidation and improved sperm quality in frozen-thawed semen of catfish [13], human [21], buffalo [6], and rooster [25]. In buffalo semen, MitoQ indicated more cryo-tolerance than Resveratrol as a cytosolic antioxidant [38].…”