An optimized centrifugation protocol for ram sperm ensuring high sample yield, quality and fertility

Area of Science:

• Reproductive Biology
• Animal Science
• Sperm Cryopreservation

Background:

• Artificial insemination (AI) in sheep requires optimized semen handling for livestock productivity.
• Sperm centrifugation is vital for AI and assisted reproductive technologies (ART), but high forces can cause sperm loss and damage.
• Optimal centrifugation conditions for ram sperm, considering extenders and forces, are not well-established, lacking in vivo fertility data.

Purpose of the Study:

• To determine ideal ram semen centrifugation conditions for maximizing sperm yield and quality.
• To ensure centrifuged ram sperm maintain unaffected fertilization ability.
• To evaluate the combined effects of extenders, cooling, and centrifugal forces on ram sperm parameters and fertility.

Main Methods:

• Experiment 1: Assessed sperm recovery and quality (motility, viability, apoptosis, mitochondrial activity) using two extenders (INRA 96, Tyrode's) and two cooling protocols (Rapid, Slow Refrigeration).
• Experiment 2: Evaluated the immediate effects of three centrifugal forces (600, 1200, 6000×g) on sperm functionality in diluted samples.
• Experiment 3: Tested the in vitro and in vivo effects of centrifugation forces on sperm quality and fertility after liquid storage.

Main Results:

• INRA 96 with Slow Refrigeration and Tyrode's at room temperature yielded highest sperm recovery and quality.
• 1200×g centrifugation resulted in the lowest pellet weight without compromising sperm viability.
• 6000×g significantly damaged sperm motility and reduced fertility, with effects persisting after storage.

Conclusions:

• Ram sperm can be safely centrifuged in INRA 96 extender at 1200×g for 10 min at 15°C.
• This protocol ensures high sperm recovery rates without detrimental effects on sperm quality and fertility.
• Optimized centrifugation is key for successful AI and enhanced sheep reproductive efficiency.