Prolonged maintenance of stallion semen by optimization of cooling conditions

Bottlenecks to the success of equine assisted reproductive technologies (ART) include suboptimal conditions for prolonged storage of stallion sperm. Shipped stallion sperm are transported in cooling devices designed to maintain temperature for up to 48 h. Increasing the storage time of cooled semen while maintaining acceptable motility would relieve logistical ART challenges. Experiments were conducted to test the hypothesis that external regulation of shipment container temperature would prolong storage time of cooled stallion semen. Initial experiments determined the effect of pre-freezing cooling cans at -20 °C or -80 °C on sperm motility. Fresh sperm was extended in INRA96 and placed in commercial Equitainers for 3.5 days (84 h). Quantification of sperm kinematics was determined every 12 h. Sperm held in Equitainers with -20°C cans maintained higher total and progressive motility than -80 °C conditions at 60 h (63 %, 29 % vs. 32 %, 17 %, respectively). Internal monitoring of Equitainers containing -20 °C freezer can temperature identified 20 °C as the threshold for rapidly decreased motility. In the second experiment, sperm were maintained in Equitainers containing -20 °C freezer cans and placed in two different conditions: 1) ambient temperature for 48 h (23 °C) or 2) 5 °C for external temperature regulation up to 8 days, or when total motility dropped below 50 %. Sperm kinematics was determined every 24 h. Total motility from samples held with external temperature regulation (5 °C) remained above 50 % for more than 7 days. These findings suggest that cooled stallion semen can be advanced beyond traditional 48 h constraints by improving temperature maintenance of storage conditions.