Controlled ice nucleation--Is it really needed for large-volume sperm cryopreservation?

Area of Science:

• Cryobiology
• Sperm Preservation
• Biotechnology

Background:

• Controlled ice nucleation (CIN) is crucial for slow freezing of large biological sample volumes to prevent cryodamage from supercooling.
• The necessity of CIN for large-volume samples using directional freezing has not been previously investigated.

Purpose of the Study:

• To test the hypothesis that a CIN stage is not required when freezing large volumes (2.5 mL) of bull semen using directional freezing.
• To evaluate the impact of omitting CIN on sperm viability, acrosome integrity, morphology, and motility.

Main Methods:

• Semen from ten bulls was cryopreserved in 2.5-mL HollowTubes using directional freezing, with and without a CIN stage.
• Post-thaw analysis included viability, acrosome integrity, normal morphology, and motility parameters via computer-aided sperm analysis.
• Sperm parameters were also assessed after 3 hours of incubation at 37°C.

Main Results:

• No significant differences were observed in any of the 29 evaluated parameters between semen frozen with and without CIN (P > 0.1).
• Sperm quality and motility were maintained similarly for both groups throughout the 3-hour incubation period.

Conclusions:

• The directional freezing technique inherently promotes continuous ice nucleation, negating the need for a separate CIN stage in large-volume semen samples.
• Omitting CIN in this context simplifies the process, reduces costs, and lowers energy consumption and carbon footprint.