Abstract
Cryopreservation is a key tool in assisted reproduction, but it often compromises post-thaw sperm quality due to cryodamage. Optimizing the initial cooling phase, specifically from room temperature to 5 °C, is a critical determinant of successful outcomes. This study aimed to evaluate the impact of different pre-freeze cooling rates on stallion sperm quality using a novel, precision cooling device. Semen samples from five healthy stallions were divided into three groups and cooled at distinct rates: Slow (0.3 °C/min), Moderate (1 °C/min), and Fast (approximately 30 °C/min). Sperm motility parameters were assessed using a Computer-Assisted Sperm Analyzer (CASA) before freezing and after thawing. Additionally, sperm integrity and physiological parameters, including viability, acrosomal integrity, Reactive Oxygen Species (ROS) expression, and mitochondrial membrane potential, were assessed by flow cytometry post-thaw. The analysis of post-thaw kinematics revealed a significant interaction between the cooling rate and processing stage (post-cooling vs. post-thaw). The Fast-cooling protocol resulted in higher post-thaw total motility (51.8%) compared to the Slow protocol (45.01%). Crucially, no significant differences were detected among cooling rates for the critical parameter of progressive motility or curvilinear velocity (VCL). Circle motility had higher values in the Fast-cooling group compared to the Slow group. Cell viability demonstrated a tendency (p = 0.08), where the Slow cooling group exhibited higher mean values (65.59%) compared to the Fast group (61.67%). Comprehensive flow cytometry assessments of other cellular integrity markers, including acrosomal integrity, mitochondrial function (MMP), and ROS expression, were statistically equivalent across all cooling rates (p > 0.05). The results confirm that this fast pre-freeze cooling rate, integrated within the highly controlled environment of Directional Freezing technology, successfully preserved essential sperm function and structure. Critically, the demonstrated functional equivalence in progressive motility validates the Fast protocol as an efficacious strategy to increase the efficiency and adaptability of equine semen cryopreservation protocols for commercial utilization.