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

Overview

• This study investigated methods to protect stallion sperm from damage caused by urine contamination, known as urospermia, by comparing simple dilution, conventional centrifugation, and colloidal density gradient centrifugation.
• They found that colloidal density gradient centrifugation best preserves sperm quality and function, especially under severe urinary contamination conditions.

Background and Importance

• Urospermia: Presence of urine in stallion ejaculate, a common cause of reduced fertility due to rapid sperm quality deterioration.
• Challenge: Urine contamination affects sperm motility, membrane integrity, and morphology, impairing fertility.
• Goal: Develop or identify semen processing methods that minimize damage from urine, improving sperm preservation and potentially fertility outcomes.

Research Objective

• Evaluate the effectiveness of three sperm processing techniques (simple dilution, conventional centrifugation, colloidal density gradient centrifugation) in preserving sperm quality in the presence of urine at varying concentrations (0%, 20%, 50%).
• Assess parameters including sperm motility, membrane integrity, morphology defects, and recovery rate after 24 hours of cooling at 5 °C.

Methods

• Subjects: Ejaculates collected from 12 stallions.
• Urine Challenges: Semen samples were artificially mixed with urine at 0%, 20%, and 50% to simulate contamination levels.
• Processing Techniques:
  • Simple dilution – semen diluted without further separation.
  • Conventional centrifugation – basic washing centrifugation to pellet cells and remove supernatant.
  • Colloidal density gradient centrifugation – uses density gradients to separate healthy sperm from debris and damaged cells.
• Storage: Processed semen samples stored at 5 °C for 24 hours to test effects of cooling on sperm viability and quality.
• Evaluations: Total motility, membrane integrity (viability), morphology defects, and sperm recovery rates were measured and compared across treatments and urine concentrations.

Key Results

• Urine Dose-Dependent Effects:
  • Increasing urine concentration caused progressive declines in sperm motility and membrane integrity.
  • Morphological defects increased with higher urine levels in all treatments.
  • Cooling for 24 hours further aggravated these negative effects.
• No Urine (0%):
  • Simple dilution maintained better sperm kinetics (motility) than conventional centrifugation.
  • Colloidal gradient centrifugation achieved the highest membrane integrity (~81.6%), indicating better sperm viability.
• Moderate Urine (20%):
  • Conventional centrifugation showed reduced total motility, highlighting susceptibility to urine damage.
• Severe Urine (50%):
  • Simple dilution and conventional centrifugation resulted in dramatic motility loss (≤10.6% after 24 hours), indicating heavy sperm damage.
  • Colloidal density gradient centrifugation better preserved motility (30.3% on average) and membrane integrity.
  • Gradient centrifugation showed fewer morphological defects.
  • Sperm recovery after gradient centrifugation was low (17.5%), indicating strong selection removing damaged/dead sperm.
  • Conventional centrifugation had high recovery rates (>82%) but retained many dead cells and debris, compromising sample quality.

Conclusions and Implications

• Colloidal density gradient centrifugation is superior for processing stallion semen contaminated with urine, especially at high contamination levels.
• This method prioritizes the quality (morphofunctional integrity) of sperm over total quantity, selecting for healthier, viable spermatozoa.
• Conventional centrifugation and simple dilution fail to adequately remove damaged sperm and contaminants under urospermia conditions.
• Using colloidal density gradients could improve semen preservation and potentially enhance fertility outcomes in stallions affected by urospermia.
• Low sperm recovery with gradients reflects discarding damaged cells, emphasizing quality control rather than maximizing cell number.