Ultra-rapid freezing in spheres yields a higher cryoresistance than in straws but remains inferior to conventional slow freezing of stallion sperm.

Overview

• This study compared different freezing methods for preserving stallion sperm, finding that ultra-rapid freezing in small spheres offers better sperm survival than freezing in straws, but neither method surpasses conventional slow freezing in effectiveness.

Research Objective

• To evaluate the cryoresistance (ability to survive freezing and thawing) of stallion sperm using three different freezing methods:
• Ultra-rapid freezing in microspheres (UR-Spheres)
• Ultra-rapid freezing in straws (UR-Straws)
• Conventional slow freezing (CS) using vapor nitrogen
• To compare the effects of these freezing methods on sperm viability, motility, structural integrity, and morphology.

Methodology

• Sampling:
• Sixteen ejaculates obtained from four healthy stallions.
• Each ejaculate divided into three equal parts for testing each freezing method.
• Freezing Methods:
• UR-Spheres: Ultra-rapid freezing by placing 30-μL droplets of sperm suspension (microspheres) directly immersed in liquid nitrogen.
• UR-Straws: Ultra-rapid freezing by direct horizontal submersion of 0.25 mL sperm-filled straws in liquid nitrogen.
• CS freezing: Conventional slow freezing by cooling the sperm in nitrogen vapor before plunging into liquid nitrogen.
• Freezing Media:
• UR methods used a medium containing 100 mM trehalose and 1% bovine serum albumin (BSA), designed for ultra-rapid freezing cryoprotection.
• CS freezing used a medium with 5% dimethylformamide, a common cryoprotectant for stallion sperm in slow freezing protocols.

Key Findings

• Conventional Slow Freezing Superior: The CS method produced significantly higher sperm cryoresistance versus both ultra-rapid methods, meaning more sperm survived and remained functional after thawing.
• UR-Spheres vs UR-Straws: Among the ultra-rapid methods, freezing in spheres resulted in improved sperm quality:
• Better sperm motility (kinematics) indicating more active swimming sperm.
• Increased viability, meaning more live sperm post-thaw.
• Greater acrosomal integrity, reflecting maintenance of a critical part of the sperm cell involved in fertilization.
• Changes in Sperm Morphology: Both ultra-rapid freezing methods caused an increase in sperm head area and perimeter compared to fresh and CS frozen sperm, which might indicate swelling or structural alterations caused by rapid freezing.

Conclusions

• The ultra-rapid freezing technique using microspheres improves sperm cryoresistance compared to ultra-rapid freezing in straws.
• However, even the best ultra-rapid freezing method tested is still inferior to the conventional slow freezing method for preserving stallion sperm quality.
• This suggests that while ultra-rapid freezing in spheres shows promise and could offer faster processing times, further optimization is needed before it can replace conventional freezing techniques in equine reproductive management.