Effect of conditioned medium from equine Wharton’s jelly mesenchymal stem/stromal cells on cool-stored stallion semen.

Overview

• This study evaluated whether conditioned medium from equine Wharton’s jelly mesenchymal stem/stromal cells (eWJ-MSC-CM) could protect cooled stallion semen during storage by assessing sperm quality parameters.
• While cooled storage reduced sperm quality over time, supplementing semen extenders with eWJ-MSC-CM did not significantly improve sperm motility, viability, or other parameters, although it was safe and showed individual stallion-dependent effects.

Background and Purpose

• Artificial insemination in horses commonly uses cooled semen transported at low temperatures (around 5°C), but stallion spermatozoa are particularly sensitive to cold stress.
• This sensitivity limits how long semen can be stored and affect fertility outcomes.
• Mesenchymal stem/stromal cells (MSCs) secrete conditioned medium (CM) containing factors that have shown protective effects on sperm in other species by improving their survival and quality.
• The study aimed to investigate if CM derived specifically from equine Wharton’s jelly MSCs (eWJ-MSC-CM) could preserve sperm quality during cooled storage in stallions, which had not been studied before.

Methods

• Five stallions with proven fertility were selected for semen collection.
• Semen samples were extended in INRA96 medium supplemented with 0% (control), 5%, or 15% eWJ-MSC-CM.
• Semen was stored at 5°C and analyzed at 0, 24, and 48 hours post-collection.
• Sperm quality assessments included:
  • Motility measured by Computer Assisted Sperm Analysis (CASA)
  • Viability (percentage of live sperm)
  • Acrosome integrity (important for fertilization capability)
  • Mitochondrial activity (as a marker of sperm function)
  • Intracellular reactive oxygen species (ROS) levels measured by flow cytometry (high ROS can damage sperm)

Key Findings

• Storage time (24 and 48 hours) caused expected declines in:
  • Sperm motility and viability decreased significantly.
  • ROS levels increased, indicating oxidative stress during cooling.
• Supplementation with eWJ-MSC-CM at either 5% or 15% concentration:
  • Did not significantly improve motility, viability, or reduce ROS compared to control.
  • Did not negatively affect sperm parameters, indicating it is safe for sperm exposure.
• There was a strong stallion-dependent effect:
  • Different stallions showed variability in semen quality and cold sensitivity.
  • Some interaction between treatment and stallion was observed for specific sperm movement characteristics and acrosome integrity, suggesting differential responses to eWJ-MSC-CM among stallions.

Conclusions and Implications

• While eWJ-MSC-CM did not enhance traditional sperm quality parameters during cooled storage, it:
  • Did not harm sperm — demonstrating safety for direct contact.
  • Exhibited some stallion-dependent effects, indicating potential for selective use or further optimization.
• Because of MSC-CM’s known anti-inflammatory and regenerative properties, even without direct motility or viability improvement, it may have value in vivo after insemination, potentially supporting the uterine environment and female fertility.
• Future research could explore these in vivo effects, different dosages, or combining MSC-CM with other protective agents to enhance cooled stallion semen preservation.