Protective Effect of Semen Extenders on Pre-Freeze Shipped Semen During Refrigeration at 5°C for 24 Hours

Effect of Semen Extenders on Pre-Freeze Shipped Semen - BotuPharma Flex Shipper and semen extenders on a lab counterRefrigerating stallion semen at 5°C for 24 hour shipment prior to freezing offers a practical field-based alternative when on-site cryopreservation is not feasible. This study evaluated the effect of semen extenders on pre-freeze shipped semen in four commercial diluents used within a passive refrigeration system to determine which best preserved sperm function before conventional freezing.

Study Design:

  • 4 stallions of proven fertility; 3 ejaculates each
  • Ejaculates divided into two groups per diluent:
    • Control: conventionally frozen immediately
    • Refrigerated: held at 5°C for 24 hours in passive refrigeration system, then frozen identically
  • Four diluents tested: Botu-Special, Botu-Semen, Botu-Semen Gold, and Botu-Turbo
  • Post-thaw assessment: total and progressive motility (computer-assisted sperm analysis), plasma membrane stability, mitochondrial potential (flow cytometry), and morphology

Key Results:

  • Botu-Semen Gold and Botu-Special refrigerated groups maintained plasma membrane stability, mitochondrial potential, total motility, progressive motility, and fast sperm percentage comparable to their respective controls
  • Botu-Semen and Botu-Turbo did not achieve satisfactory results and were excluded from final analysis
  • Sperm morphology showed no significant differences across any group
  • No negative impact on mitochondrial function was detected in the successful diluent groups

Conclusions:

  • Botu-Special and Botu-Semen Gold successfully preserve sperm function during 24-hour pre-freeze refrigeration at 5°C
  • This technique offers a viable field-based strategy for veterinarians working in locations without immediate cryopreservation capability
  • Diluent selection is critical and there is a potential for positive or negative effect of semen extenders on pre-Freeze shipped semen — not all commercial media are suitable for pre-freeze refrigeration protocols

(Zanlorenzi-Silva LP, Freitas-Dell’Aqua CP, Baby MEA, Trinque CM, Frasson MS, Canuto LEF, Alvarenga MA, Dell’Aqua JR JA, Cavalero TMS, Papa FO. The protective capacity of different diluents on stallion sperm during storage in a passive refrigeration system “BotuflexⓇ” for 24 h at 5°C before freezing. JEVS 145:105337 – Presented in Association with and by Permission of the International Society for Equine Reproduction)