Apoptotic-like changes of boar spermatozoa in freezing media supplemented with different antioxidants

Abstract

This study evaluated the effect of supplementing the freezing extender with exogenous antioxidants on apoptotic-like changes in post-thaw boar spermatozoa. A total of 36 ejaculates were resuspended in standard lactose-egg yolk-glycerol extender supplemented with antioxidant to final concentrations of 0 (as control), 2.5mM GSH (group I), 5.0 mM GSH (group II), 150 IU/mL SOD (group III), 300 IU/mL SOD (group IV), 200 IU/mL CAT (group V), 400 IU/mL CAT (group VI), 150 IU/mL SOD+200 IU/mL CAT (group VII), 300 IU/mL SOD+400 IU/mL CAT (group VIII). Sperm motility and apoptotic-like changes were determined before and after freeze-thawing. The various markers of apoptotic-like changes were measured: plasma membrane permeability by YO-PRO-1/PI assay, phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V, mitochondrial transmembrane potential detected by JC-1, and DNA fragmentation evaluated by TUNEL assay. The highest percentage of progressive motile sperm was noticed in group II (PM% 64.2±15.4) compared with control (PM% 36.8±5.5). The supplementation of 400 IU/mL CAT (group VI) revealed significant (P<0.01) reduction of apoptotic-like changes (YO-PRO-1 + /PI -: 13.1±7.5%, AnV + /PI -: 9.9±4.1%) in frozen-thawed spermatozoa compared with extender supplemented with 200 IU/mL CAT (group V). Irrespective of the concentration used, SOD and CAT in combination (group VII and group VIII) significantly (P<0.01) improved post-thaw sperm survival compared with the control. Evaluation by TUNEL assay revealed that cryopreservation and thawing did not induce DNA fragmentation in boar spermatozoa.

A new sperm selection criterion for cryopreservation of boar semen

“…The stability and permeability of the sperm membrane are closely related to its functionality, and thus evaluation of membrane permeability elucidates its organization and its capability to undergo biophysical changes such as water crystallizing and the movement of cryoprotectant agents during cryopreservation (Hossain et al, 2011). YO-PRO-1 is an impermeable membrane probe that can leak in after destabilization of the membrane and it is used for detecting early membrane permeability (Trzcińska et al, 2015b). The structure of the sperm cell membrane and the processes involved in the acrosome reaction are significant for fertilization.…”

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

“…The Vybrant Apoptosis Assay Kit #4 (Molecular Probes Inc., Eugene, OR, USA) was used to assess the percentages of viable and plasma membrane apoptotic‐like changes in boar spermatozoa, according to a previously described method (Martin, Sabido, Durand, & Levz, ), with some modifications (Trzcińska & Bryła, ). Briefly, 1 μl YO‐PRO‐1 solution (100 μM) and 1 μl PI (2 μM) were added to 1 ml of sperm suspension (4 × 10 6 spermatozoa/ml) and incubated for 15 min at 37°C.…”

The roles of reactive oxygen species and antioxidants in cryopreservation

“…However, when the same dose of SOD was co-administered with catalase, significant improvement in oocyte survivability was observed [124]. For the case of sperm cryopreservation, supplementation of SOD alone has no effect on the general sperm parameters such as motility (total and progressive motility), viability and percentage of sperm with high MMP, while supplementation of catalase alone was beneficial only at high concentrations [244]. Notably, when catalase and SOD were used in combination, sperm parameters such as total motility was greatly improved as compared with individual use of them at the respective dose [244].…”

“…For the case of sperm cryopreservation, supplementation of SOD alone has no effect on the general sperm parameters such as motility (total and progressive motility), viability and percentage of sperm with high MMP, while supplementation of catalase alone was beneficial only at high concentrations [244]. Notably, when catalase and SOD were used in combination, sperm parameters such as total motility was greatly improved as compared with individual use of them at the respective dose [244]. In another example, sperm cells frozen in SOD and catalase, or vitamins C and E led to significantly improved parameters such as reduced ROS [215] and increased lateral head displacement of the sperm cells [245] whereas previously such parameters were not improved when the antioxidants were used individually [215,245].…”