Proteomic analysis reveals the potential positive effects of Mito-TEMPO on ram sperm motility and fertility during cryopreservation.

The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT40 groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT40 group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.

Shi L ，Shi J ，Feng J ，Zhang P ，Ren Y ... -  《-》

Effects of mitoquinone (MitoQ) supplementation during boar semen cryopreservation on sperm quality, antioxidant status and mitochondrial proteomics.

Mitochondria are the most important organelles and the main reactive oxygen species producers in spermatozoa. The objective of this study was to investigate the effects of mitochondria-targeted antioxidant mitoquinone (MitoQ) supplementation in boar semen extender during cryopreservation on sperm quality, antioxidant status and the changes of sperm mitochondrial proteomic profile. Semen collected from 10 Large White boars was cryopreserved in lactose-egg yolk extender supplemented with various concentrations of MitoQ (0, 5, 10, 20 and 40 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The comprehensive mitochondrial proteomic profiling was performed on spermatozoa in the control and MitoQ10 groups. Supplementation with 10 μM of MitoQ resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed boar sperm were also elevated in the MitoQ10 group. Excessive MitoQ (40 μM) supplementation induced a reduction of sperm motility parameters, sperm quality and antioxidant status and the abundance of GLUT3 and 8 proteins. A total of 189 proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05, and 33 of them were dramatically (FC > 1.5) regulated by MitoQ. These DEPs are mainly involved in sperm motility, energy metabolism and oxidative phosphorylation. Our data suggest that the beneficial effect of MitoQ on cryopreserved boar semen is achieved by regulating sperm antioxidant capacity and the mitochondrial proteins related to motility and metabolism.

Shi L ，Zhang Y ，Huang X ，Shi M ，Sun D ，Zhang Y ，Li W ，Jin T ，Feng J ，Xing J ，Li B ，Cao G ... -  《-》

Effects of freezing extender supplementation with mitochondria-targeted antioxidant Mito-TEMPO on frozen-thawed rooster semen quality and reproductive performance.

Rooster semen cryopreservation is a useful method to utilize semen samples for artificial insemination in commercial flocks, but with use of the freezing-thawing process there is a reduction in the quality and fertilization capacity of rooster spermatozoa post-thawing. The aim of the current study was to investigate the efficacy of the mitochondria-targeted antioxidant Mito-TEMPO on rooster sperm quality and fertilization capacity after conducting the freezing-thawing processes. Semen samples were diluted and there were five equal aliquots supplemented with 0, 0.5, 5, 50 and 500 μM Mito-TEMPO. Semen samples were subsequently cryopreserved in liquid nitrogen. After thawing, sperm motility, lipid peroxidation, membrane functionality, normal morphology, mitochondria active potential, acrosome integrity, viability, apoptotic-like changes, DNA fragmentation, hydrogen peroxide concentration and fertilizing capacity were evaluated. Supplementation of Lake medium with 5 and 50 μM Mito-TEMPO resulted in greater (P ≤ 0.05) total sperm motility, progressive motility, average path velocity, membrane functionality, mitochondria active potential, acrosome integrity and viability compared with semen of the other groups. Lipid peroxidation, late apoptotic-like changes, DNA fragmentation and hydrogen peroxide content, however, were less (P ≤0.05) in semen samples supplemented with 5 and 50 μM Mito-TEMPO compared to other groups. Furthermore, fertility percentages were greater when there was supplementation with 5 and 50 μM Mito-TEMPO compared to the control group. Mitochondria-targeted antioxidant Mito-TEMPO could be included in semen extender before cryopreservation to improve quality and fertilization capacity of rooster semen after thawing of cryopreserved samples.

Masoudi R ，Asadzadeh N ，Sharafi M 《-》

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p < 0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p < 0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Kumar A ，Kumar Ghosh S ，Katiyar R ，Gemeda AE ，Rautela R ，Bisla A ，Srivastava N ，Kumar Bhure S ，Devi HL ，Chandra V ... -  《-》

Supplementation of ram's semen extender with Mito-TEMPO II: Quality evaluation and flow cytometry study of post-thawed spermatozoa.

Cryopreservation is an effective method to spread qualified ram spermatozoa for reproductive goals in different farms, but cryopreservation's shocks reduce sperm quality. This study investigated the efficacy of the new mitochondria-targeted antioxidant Mito-TEMPO on post-thawed quality of spermatozoa in sheep. Collected samples were divided into five groups and after dilution, received different doses of Mito-TEMPO (0, 0.5, 5, 50 and 500 µM), and frozen. Thawed sperm motility parameters, malondialdehyde content, membrane functionality, abnormal morphology, mitochondria activity, acrosome integrity, DNA fragmentation, ROS concentration, viability and apoptotic-like changes, were evaluated. According to the results, Mito-TEMPO (5 and 50 μM) improved (p ≤ 0.05) motility parameters, average path velocity, membrane functionality, mitochondria activity and viability compared with the other groups. Moreover, apoptotic-like changes, lipid peroxidation and ROS concentration were lower (p ≤ 0.05) in groups received 5 and 50 μM Mito-TEMPO. Mito-TEMPO showed no effect (p > 0.05) on sperm acrosome integrity, morphology and DNA fragmentation. In conclusion, Mito-TEMPO as a targeted antioxidant could be an efficient cryo-additive to enhance quality parameters of post-thawed ram semen.

Zarei F ，Daghigh-Kia H ，Masoudi R 《-》