Evaluating stallion sperm quality post-thaw: Effects of selection methods on concentration, motility, and mitochondrial activity

The commercial use of frozen-thawed equine semen for foal production is a well-established clinical practice. However, to address infertility issues in some stallions, sperm selection techniques are essential for improving semen quality. This study aimed to evaluate the quality of post-thaw sperm processed using either a density gradient (EquiPure; NidaCon International AB, Sweden) or a microfluidic device (VetMotl; VetMotl Inc., Gaithersburg, MD, USA). Frozen-thawed semen from 6 stallions was analyzed with 2 replicates per stallion. Thawed straws were diluted 1:2.5 with Botusemen® Gold (Botupharma USA, Phoenix, AZ, USA) and aliquoted into their respective selection methods (control, EquiPure, or VetMotl) for processing. The thawed sperm were assessed for concentration (CONC; 10⁶/mL), total motility (TMOT), progressive motility (PMOT), and active mitochondria measured by double-stain fluorescence assay (H33342/Rhodamin123) using a computer-assisted sperm analyzer (CASA; AndroVision®, Minitube International, Verona, WI, USA). Statistical analysis was performed using the mixed model of SAS V9.4, with stallion included as a random effect to account for variability between individual stallions. Statistical significance was set at P < 0.05, and trends noted at P < 0.10. A significant effect on mean CONC was observed (P = 0.0004). VetMotl exhibited a significantly lower mean CONC (70.925 ± 77) compared with both the control (350.05 ± 77) and EquiPure (266.39 ± 77). There was no difference in TMOT between processing methods (P = 0.1118). Interestingly, an overall difference was observed for PMOT (P = 0.0038). The control method had a lower mean percentage of PMOT (38.5 ± 7) compared with both EquiPure (49.5 ± 7) and VetMotl (56.7 ± 6). However, no difference in PMOT was observed between EquiPure and VetMotl (P = 0.1226). Furthermore, there was a difference in active mitochondria (P = 0.0003). VetMotl had a greater mean percentage of active mitochondria (40.5 ± 7) compared with both the EquiPure (15.8 ± 3; P < 0.0001) and the control (23 ± 3; P = 0.0012). Overall, this study highlights the differential impact of sperm selection methods on the quality of post-thaw equine spermatozoa. While VetMotl showed advantages in terms of active mitochondria, it did not outperform EquiPure in PMOT. These findings suggest that both methods have distinct benefits and may be utilized based on specific criteria for improving semen quality in equine reproductive practices.