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 《-》

Preservation of the quality and fertility potential of post-thawed rooster sperm using MitoQ.

Cryopreservation of rooster spermatozoa is an efficient procedure to spread qualified semen samples for reproductive goals in commercial flocks, but the freeze-thawing process reduces the quality and fertility potential of post-thawed sperm cells. This study was aimed to investigate the effect of the mitochondria-targeted antioxidant MitoQ on rooster sperm quality and fertility potential preservation during freeze-thawing process. Semen samples were collected and diluted in the Lake medium, assigned into five equal aliquots, supplemented with 0, 1, 10, 100 and 1000 nM MitoQ, and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondria active potential, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration and fertility potential were evaluated. According to the results, freezing extender supplementation with 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, membrane functionality, mitochondria active potential, acrosome integrity and viability compared to the other groups. On the other hand, lipid peroxidation, apoptosis rate, DNA fragmentation and ROS concentration were lower (P ≤ 0.05) in groups received 10 and 100 nM MitoQ compared to other groups. Moreover, fertility rate was higher in groups received 10 and 100 nM MitoQ compared to control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in rooster and it could be an effective additive for supplementation of rooster's semen cryopreservation medium during reproductive programs.

Alipour-Jenaghard P ，Daghigh-Kia H ，Masoudi R 《-》

The mitochondria-targeted antioxidant Mito-TEMPO conserves rooster's cooled semen quality and fertility potential.

The PUFAs content of rooster sperm cells makes them vulnerable to the thermal shocks during chilling storage, which reduces the fertility performance of cooled sperm. Extender supplementation with antioxidants is a reasonable method to conserve sperm fertility potential during cooling storage process. The aim of this study was to determine the effect of Mito-TEMPO addition to the Lake medium on rooster sperm quality and fertility potential during cooling process. Semen samples were diluted in the Lake medium and assigned into five equal aliquots and supplemented with 0, 0.5, 5, 50 and 500 μM Mito-TEMPO. Then, the samples were cooled at 5 °C and conserved up to 50 h. Total motility, progressive motility, morphology, viability, membrane integrity, lipid peroxidation and mitochondrial activity of samples were analyzed during 0, 25 and 50 h of cooling period. Artificial insemination was also conducted using 25 h-cooled semen. No significant difference was observed among different treatments during quality evaluations at 0 h storage. Extender supplementation with 5 and 50 μM Mito-TEMPO presented greater (P ≤ 0.05) total motility, progressive motility, viability, membrane integrity and lower lipid peroxidation compared to other groups during 25 and 50 h cooling storage. Mitochondrial activity was higher (P ≤ 0.05) in groups received 5, 50 and 500 μM Mito-TEMPO than others. Fertility rate of 25 h-cooled-stored samples was higher (P ≤ 0.05) in groups containing 5 and 50 μM Mito-TEMPO compared to control group. In conclusion, addition of 5 and 50 μM Mito-TEMPO as a mitochondria-targeted antioxidant to the storage medium could be a suitable method to conserve rooster semen quality against stressful conditions of cooling storage process.

Masoudi R ，Asadzadeh N ，Sharafi M 《-》

L-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm.

Rooster semen cryopreservation is not efficient for artificial insemination in breeder flocks. L-Carnitine (LC) has been evaluated for effectiveness in cryopreservation media on the characteristics of rooster sperm after freeze-thawing. Motility characteristics, membrane functionality, abnormal morphology, apoptotic like changes, mitochondria activity and lipid peroxidation of rooster sperms were assessed after freeze-thawing with different concentrations of LC in Beltsville medium. Semen samples were collected from 12 roosters, twice a week, and diluted in the extenders that contained different concentrations of LC. Supplementation of Beltsevile with 1 and 2 mM LC was found to result in higher total motility (68.2± 1.7% and 69.1± 1.7%, respectively), progressive motility (28.4± 1.6%, 29.8± 1.6%), membrane functionality (76.2± 1.9% and 75.9± 1.9%), viability (58.2 ± 1.1%, 59.1 ± 1.1%) and lower significant of lipid peroxidation (2.53 ± 0.08 nmol/ml, 2.49 ± 0.08 nmol/ml) compared to control group containing no LC. Lower motility, progressive motility, and viability were observed in frozen-thawed sperm in extender containing 8 mM LC (35.8± 1.7%, 9.6± 1.2% and 27.1 ± 1.2%, respectively) compared to control. Morphology and mitochondrial activity were not affected by different concentrations of LC. Our results showed that supplementation of Beltsville extender with 1 and 2 mM LC significantly improved the quality of rooster sperm quality after freeze-thawing.

Fattah A ，Sharafi M ，Masoudi R ，Shahverdi A ，Esmaeili V ，Najafi A ... -  《-》

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 《-》