Semen cryopreservation is critical for appropriate planning of both AI and IVF trials, improving the benefit:cost ratio. However, cryopreservation induces damage in mammalian spermatozoa, resulting in decreased fertility (Medeiros et al. 2002 Theriogenology 57, 327-344). It is known that cryopreservation and thawing induce apoptosis in a variety of cells, including bovine sperm (Anzar et al. 2002 Biol. Reprod. 66, 354-360). Cryotolerance of in vitro-produced bovine embryos was recently improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD-FMK), during vitrification and subsequent culture (Pero et al. 2018 Theriogenology 108, 127-135). The aim of this work was to evaluate whether treatment of bovine frozen-thawed sperm with the caspase inhibitor Z-VAD-FMK may prevent aberrant apoptosis and consequently improve sperm viability. Six ejaculates from 12 bulls were used for the trial. Semen was diluted at 37°C with BioXcell extender (BioXcell, West Lebanon, NH) to a final concentration of 30×106 spermatozoa mL−1, and straws were kept at 4°C for 4h and then frozen in an automated system. After thawing, Percoll-separated spermatozoa were incubated at 37°C for 1h with 0, 20, and 100µM ZVAD-FMK. Sperm viability and membrane integrity were assessed by Trypan Blue/Giemsa and hypo-osmotic swelling test, respectively, as previously described (Longobardi et al. 2017 Theriogenology 88, 1-8). Sperm motility was examined by phase contrast microscopy at 40× magnification on a thermoregulated stage at 37°C. Apoptosis was evaluated by TUNEL technique, which assesses DNA fragmentation (Takeda et al. 201561, 185-190). The mitochondrial membrane potential was then assessed by flow cytometric analysis with the mitochondrial probe JC-1 (Garner and Thomas 1999Mol. Reprod. Dev. 53, 222-229). Data were analysed by ANOVA using least significant difference as post-hoc test. The treatment of bovine frozen-thawed sperm with 100µM ZVAD-FMK decreased the percentage of sperm exhibiting DNA fragmentation (17.8±1.1, 13.3±2.8, and 10.5±2.5 with 0, 20, and 100µM ZVAD, respectively; P<0.05). Moreover, both concentrations of ZVAD-FMK increased the percentage of hypo-osmotic swelling test+ sperm, indicating improved membrane integrity compared with the control (60.5±3.5, 70.9±2.1, and 74.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively; P<0.01). However, no differences were found in sperm viability (82.3±0.5, 84.6±1.0, and 84.3±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively) and motility (60.0±2.9, 62.5±3.4, and 67.5±2.1 with 0, 20, and 100µM ZVAD-FMK, respectively). Furthermore, no differences were observed among groups in the percentage of sperm exhibiting normal mitochondrial membrane potential (62.4±12.7, 57.9±12.8, and 50.8±8.8 with 0, 20, and 100µM ZVAD, respectively). In conclusion, caspase inhibition with 100µM ZVAD-FMK after thawing was effective in reducing sperm DNA fragmentation and increasing sperm membrane integrity, suggesting a beneficial effect on fertility. However, as the other fertility-related parameters did not improve, further investigations are required to draw definite conclusions.
A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples