Cryopreservation of flow-sorted bovine spermatozoa

Area of Science:

• Reproductive biology
• Animal science
• Biotechnology

Background:

• Flow cytometry sorting and cryopreservation are critical for sexed sperm technologies.
• Maintaining sperm viability after these processes is essential for practical applications like artificial insemination.

Purpose of the Study:

• To optimize protocols for maximizing sperm viability after flow cytometry sorting and subsequent cryopreservation.
• To assess the impact of various experimental parameters on post-thaw sperm motility and integrity.

Main Methods:

• Sperm were stained with Hoechst 33342 dye and interrogated with laser light.
• Post-sort sperm concentration was achieved via centrifugation.
• Sperm motility was assessed after thawing following different extension media, equilibration times, and laser powers.

Main Results:

• Concentrating sperm to 10-20 x 10(6)/ml minimized dilution effects.
• Lower laser power (100 mW) resulted in better post-thaw motility than higher power (150 mW).
• TRIS-based medium and shorter equilibration times (3-6 hours at 5°C) improved post-thaw motility compared to EYC, TEST media, or 18-hour equilibration.

Conclusions:

• Current flow cytometry sexing causes minor damage to sperm, less than routine cryopreservation.
• Optimized procedures improve sperm viability and fertilizing capacity.
• Sexed bovine sperm for commercial artificial insemination are anticipated within two years.