Effect of caspase inhibitor Z-VAD-FMK on bovine sperm cryotolerance

Reprod Domest Anim. 2020 Apr;55(4):530-536. doi: 10.1111/rda.13648. Epub 2020 Feb 17.


The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 hr with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (p < .05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4, respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (p < .05), while the percentage of spermatozoa with high ΔΨm increased (p < .05). The results of Experiment 2 showed that 1-hr treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (p < .05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (p < .05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.

Keywords: apoptosis; bovine semen; caspase inhibitor.

MeSH terms

  • Amino Acid Chloromethyl Ketones / pharmacology*
  • Animals
  • Caspase Inhibitors / pharmacology*
  • Cattle
  • Cell Survival
  • Cryopreservation / methods
  • Cryopreservation / veterinary
  • DNA Fragmentation
  • Freezing
  • Male
  • Semen Preservation / methods
  • Semen Preservation / veterinary
  • Sperm Motility
  • Spermatozoa / drug effects*
  • Spermatozoa / physiology


  • Amino Acid Chloromethyl Ketones
  • Caspase Inhibitors
  • benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone