Apoptotic-like changes in equine spermatozoa separated by density-gradient centrifugation or after cryopreservation.

This study aims to examine the markers of apoptosis (programmed-cell death) in horse sperm after undergoing density-gradient centrifugation and cryopreservation. The results suggest that such processes can lead to apoptotic-like changes in the spermatozoa, with such changes being more prevalent following cryopreservation.

Research Methods

• Density-gradient centrifugation and cryopreservation steps were applied to split sperms into high and low density subgroups.
• Markers for apoptosis such as the percentage of live sperm, caspase (an enzyme involved in programmed-cell death) activation, mitochondrial membrane potential (MMP), plasma membrane permeability, and DNA fragmentation were compared between these subgroups.
• Motility of the sperm was also evaluated by comparing total motility and progressive motility (ability of sperm to move forward) between high-density and low-density subgroups.
• Special attention was given to the externalization of phosphatidylserine (a marker for early apoptosis) between the different groups.

Findings

• The findings highlighted a significant difference in the markers of apoptosis between the high-density and low-density sets of sperm after density-gradient centrifugation. This difference was further amplified after cryopreservation.
• While high-density sperm samples showed less presence of apoptotic markers and greater motility, the low-density sperm demonstrated more apoptotic signs and less motility.
• Interestingly, cryopreservation led to an increase in the percentage of active caspases, indication of low MMP, altered plasma membrane permeability, and decreased total and progressive motility.
• However, cryopreservation did not significantly affect the DNA integrity of the cells as the percentage of DNA fragmented cells remained relatively stable before and after the process.
• Regardless of the processes, detectable apoptotic-like changes were observed in ejaculated equine spermatozoa. These changes, however, were more prominent after cryopreservation.

Conclusion

The study concluded that apoptotic-like changes occurred in equine sperm after undergoing density-gradient centrifugation and cryopreservation. Notably, cryopreservation was particularly impactful, triggering more signs of apoptosis. However, the DNA integrity appeared resilient to these changes, remaining largely undamaged after these procedures. The information obtained from this research might be useful in enhancing sperm preservation techniques.