Addition of cholesterol linked to cyclodextrin in processing of cryopreserved equine spermatozoa and addition of motility stimulants post-thaw.

Overview

• This research examined how adding cholesterol linked to cyclodextrin (CLC) before freezing and using a motility stimulant called FertTalp (FT) after thawing affects the quality and performance of frozen and thawed stallion sperm.
• The goal was to improve sperm motility and function after cryopreservation, which is important for equine breeding and artificial insemination.

Background and Objectives

• Cryopreservation (freezing) of stallion sperm often reduces its motility and viability after thawing, limiting fertility success.
• Cholesterol is known to stabilize sperm membranes during freezing, potentially protecting sperm from damage.
• Cyclodextrin molecules can deliver cholesterol to sperm membranes effectively.
• FertTalp (FT) is a solution designed to stimulate sperm motility after thawing.
• This study’s main aim was to:
• Investigate the effects of different doses of cholesterol-loaded cyclodextrin (CLC) added before freezing on sperm motility and membrane integrity.
• Evaluate if adding FT after thawing could further improve sperm motility and longevity.

Methods

• Sample Collection:
  • Sperm was collected from 12 stallions, with two ejaculates each.
  • Each ejaculate was divided into four treatment groups based on CLC dose: 0 mg (control), 1.0 mg, 1.5 mg, and 2.0 mg.
• Cryopreservation:
  • Sperm was frozen with the assigned CLC doses prior to freezing.
• Post-Thaw Evaluation:
  • Sperm motility and movement characteristics were analyzed using computer-assisted sperm analysis (CASA).
  • Flow cytometry was used to assess mitochondrial membrane potential and membrane lipid disorder, indicators of sperm health.
• Additional Testing:
  • FertTalp (FT) was added to some samples after thawing to examine its effect on motility up to 120 minutes post-thaw.

Key Findings – Experiment I (CLC Supplementation)

• The group treated with 1.5 mg of CLC (G3) showed significantly better total motility and progressive motility immediately after thawing compared to control:
• Total motility: 64.9% vs 55.5% in control
• Progressive motility: 40.5% vs 31.9% in control
• There were no significant differences in mitochondrial potential or membrane lipid disorder among groups, indicating no negative impacts on sperm viability or membrane condition.
• During a thermoresistance test (testing longevity at elevated temperatures), cholesterol supplementation did not improve overall survival time.
• However, G3 maintained higher velocity parameters (speed and movement paths) compared to some other doses (such as G2), suggesting better sperm movement quality over time.

Key Findings – Experiment II (Post-Thaw FertTalp Use)

• FT did not improve sperm motility in the first 40 minutes after thawing.
• At 80 and 120 minutes post-thaw, FT-treated samples exhibited:
• Higher proportions of rapid-moving sperm.
• Increased straight-line velocity (VSL) and average path velocity (VAP), indicating quicker and more efficient movement.
• Greater linearity in movement, meaning sperm swam straighter paths which is favorable for fertilization.
• FT increased the proportion of viable sperm that did not contain intracellular hydrogen peroxide (H₂O₂), suggesting a slight reduction in oxidative stress.
• However, this antioxidative benefit was relatively small.

Conclusions and Practical Implications

• Adding 1.5 mg of cholesterol-loaded cyclodextrin before freezing improves the initial motility of thawed equine sperm without compromising mitochondrial function or membrane integrity.
• Using FertTalp after thawing enhances sperm velocity and supports prolonged functional activity, extending the period sperm remains motile and competent.
• The combined approach—cholesterol supplementation before freezing and motility stimulant afterward—may offer a practical way to optimize freezing protocols and improve fertility outcomes in stallion semen cryopreservation.
• These findings can help equine reproductive specialists improve artificial insemination success rates by increasing the number of motile, functionally competent sperm after thawing.