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.
Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry
