Effect of diluent composition on the dynamics of sperm DNA fragmentation and other sperm quality parameters in ram during incubation at 37 °C

- The results of dynamic SDF are highly dependent on the diluent employed.
- Some diluents are not recommended to study male susceptibility to SDF.
- Some diluents, such as MOPS, may be useful for storage of ram semen.
- The pH of the diluents is correlated to sperm DNA fragmentation in ram.

This study was conducted to assess the effect of different semen extenders on the dynamics of sperm DNA fragmentation (SDF) and other quality sperm parameters in ram. To this end, second ejaculates from 6 adult males were collected using an artificial vagina and diluted in either INRA-96®, milk, MOPS, TRIS, TES, HEPES, citrate or phosphate-based extenders. Semen samples were incubated at 37 °C and the SDF, motility and membrane integrity were assessed after 0, 3, 6 and 24 h intervals. Significantly higher SDF indexes were obtained at 6 and 24 h of incubation in the milk-based than in all the buffer-based extenders. At 24 h, sperm diluted in INRA also reached a higher SDF than MOPS, TRIS, citrate, and phosphate-based extenders. Diluent composition also affected sperm motility and membrane integrity parameters. The diluent had a clear impact on the pH of the samples, with a significant drop at 6 h of incubation in the samples diluted in INRA, milk and citrate-based diluents, but only at the end of the incubation period (24 h) in the other diluents. A significant negative correlation was observed between the pH of the media and SDF (−0.67). It was concluded that the results of the dynamics of sperm DNA fragmentation are highly dependent on the diluent employed during incubation at 37 °C, and this should be taken into account in studies evaluating this parameter. The use of some diluents would reduce, or even prevent, sperm DNA fragmentation during incubation, so that possible differences in susceptibility between males would be masked.