Density gradient centrifugation preserves sperm kinetics and morphology in stallion ejaculates with severe urospermia

Urospermia is a frequent cause of subfertility in stallions due to the rapid deterioration of sperm quality.

This study aimed to evaluate the effectiveness of different processing methods in minimizing the deleterious effects of urine on cooled equine semen.

Ejaculates from 12 stallions were subjected to simple dilution, conventional centrifugation, and colloidal density gradient centrifugation, under challenges of 0%, 20%, and 50% urine, and stored at 5 °C for 24 h.

A severe dose-dependent effect was observed: increasing urinary concentration progressively reduced sperm kinetics and membrane integrity, while increasing the incidence of morphological defects in all treatments, with further deterioration after cooling. In the absence of urine, the diluted group showed superior kinetics compared with the centrifuged group, whereas the gradient group achieved higher membrane integrity (81.6 ± 10.0%). Under moderate challenge (20%), total motility was reduced in the conventional centrifugation group, highlighting the limitations of this method. At the 50% concentration, dilution and conventional centrifugation showed a severe decline in total motility (motility ≤ 10.6% after 24 h). In contrast, the gradient treatment maintained motility (30.3 ± 19.8%) and cellular integrity, and showed a lower prevalence of total defects. Sperm recovery in the gradient treatment decreased to 17.5 ± 8.8% under 50% urine, indicating strong selection against damaged cells, whereas conventional centrifugation retained dead cells and debris (>82% recovery).

In conclusion, colloidal density gradient centrifugation is the most effective strategy under severe urospermia, prioritizing morphofunctional integrity over absolute numerical recovery.