This study was carried out to investigate the effect of the semen freeze–thawing process on the functionality and molecular structure of ram spermatozoa. The temperature of pooled and diluted semen at 38°C (group 1, control) was lowered to 5°C (group 2), and it was subjected to glycerolisation–equilibration (group 3), frozen and thawed (group 4). Compared to the control, deterioration in spermatological parameters and significant increases in lipid peroxidation and global DNA methylation levels were observed in groups 3 and 4. When compared with the control, significant downregulation in the levels of miR-485 of group 2, miR-29a of group 3 and let-7a, miR-485 and miR-29a of group 4, and significant upregulation in the levels of miR-107 of group 3 and miR-127 of groups 3 and 4 were detected. In comparison to the control, significant upregulation in the levels of CatSper1, CatSper2, CatSper3, CatSper4, ANO1 and TRPM3 of group 2, CatSper4, ANO1 and TRPM3 of group 3 and KCNJ11 of group 4, and significant downregulation in the CatSper 3 level of group 4 were determined. As a result, the semen freeze–thawing process causes motility and morphological disorders in rams. This may be due to molecular changes associated with lipid peroxidation in spermatozoa.

The present study investigates the changes in M1/M2 macrophage polarization resulting from unilateral testicular torsion in the bilateral testis. The study sample included 63 male Sprague–Dawley rats, which were randomly divided into nine groups (n = 7): Control, Sham (4 h (4 h), 24 h, 7 days (7d), 14d), and Torsion/Detorsion (T/D) (4 h, 24 h, 7d, 14d). Histopathological evaluations revealed no changes in the Sham groups, while T/D was noted to cause edema, vascular occlusion, disruption of seminiferous tubule epithelial organization, germ cell abnormalities and structural anomalies in the experimental rats, the severity and extent of which increased from 4 h to 14d after T/D. The Cosentino scores used to determine the degree of histological damage were consistent with the histopathological findings in all groups, while the Johnsen scores, as a marker of spermatogenesis, were lower in the T/D groups. Seminiferous tubule diameters and germinal epithelial thickness decreased significantly in parallel with increased tubule damage in the ipsilateral testicles. Testicular torsion significantly affected sperm motility, with significant reductions observed in the T/D 7d and T/D 14d groups. A hormone profile analysis revealed decreased testosterone levels in both the Sham and T/D groups when compared to the Controls. CD68 and CD163 immunoreactivities, as M1 and M2 macrophage surface markers, were determined in the testicular tissue using the avidin–biotin-peroxidase complex method. T/D interventions caused M1/M2 macrophage polarization changes and increased M1 macrophages, particularly in contralateral testicular tissue. The increase in M1 macrophages in contralateral testicular tissue following T/D in the present study suggests that cell processes, including macrophages, may play an important role in contralateral testicular injury.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C₆₀HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 μM C₆₀HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at −140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 μM C₆₀HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C₆₀HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 μM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 μM doses) and catalase (between 1 and 40 μM doses) activities. In conclusion, results of this study show that the C₆₀HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze–thawing.