Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry.

The study presents a method that uses double-staining for testing the health and mitochondrial function of stallion sperm using flow cytometry. This technique provides a reliable and quick assessment of stallion semen.

Research Methodology and Findings

• The research developed a double-staining method to evaluate the viability and function of mitochondria in stallion sperm. The viability of the sperm was checked with propidium iodide (PI) exclusion, while the functionality of mitochondria was tested using the intensity of rhodamine 123 (R123) fluorescence.
• The study found that estimates of sperm viability measured by PI were equivalent to those made using Hoechst 33258 staining and fluorescent microscopy.
• The double staining with PI and R123 was validated by combining viable spermatozoa with different levels of freeze-shocked (membrane-damaged) cells. There was a high correlation between the number of membrane-damaged sperm added and the increase in dead cells stained positively with PI.
• The research showed that optimal mitochondrial activity (OMA), as evaluated by R123 intake, was also reduced by the percentage of added membrane-damaged cells.

Experimentation with Mitochondrial Inhibitors

• Mitochondrial inhibitors rotenone and monensin significantly reduced optimal mitochondrial activity. There was a correlation between the amount of inhibitors added and the population of sperm cells showing minimum R123 staining, indicating reduced OMA.
• Equivalent proportions of membrane-damaged cells led to similar results around viability and mitochondrial activity. These results were consistent irrespective of whether single or double staining was used.

Implication of Findings

• The double-staining method revealed a significant correlation between viability (PI-negative) sperm cells and spermatozoa with optimally functioning mitochondria.
• The flow cytometric analyses using double-staining offered a quick (10,000 cells/minute) and reliable assessment of stallion semen quality.

The research provides valuable insights for improving methods in sperm health assessment, particularly for livestock and other animals where breeding efficiency is critical. Future studies may expand the applications of this method to other animal species.