Effect of bovine seminal plasma Binder of Sperm Protein 1 on stallion sperm cryopreservation

Proteins present in seminal plasma directly interact with the plasma membrane of spermatozoa, being important for successful sperm maturation and fertilization. The aim of this work was to evaluate the effect of Binder of Sperm Protein 1 (BSP1) from the seminal vesicle of bulls on semen quality of stallions and its effect on freezability of this semen. The BSP1 protein was isolated using the ammonium sulfate technique and lyophilized. Protein presence was observed at the highest concentration of BSP1 (98%) confirmed by SDS-PAGE and Western blot. The effect of addition of BSP1 to stallion semen was tested at concentrations 0 (control), 10, 20 and 40 μg/mL by addition to a commercial freezing extender. Semen from 4 stallions with 5 ejaculates per stallions was used and cryopreserved using a programmable machine (TK 3000®). After thawing (37°C / 30 seconds), samples were submitted to sperm kinematic analysis by CASA (time 0 hour and Thermo Resistance Test - TRT of 1 hour at 37°C), integrity of plasma membranes (iMP) and acrosomal membrane integrity (iAC; epifluorescence microscopy. The percentage values were subjected to arcsin transformation and means were compared using T-test at 5% probability comparing time 0 vs. TRT of 1 hour. No difference was observed for iMP andiAC regardless of BSP1 concentration. At time 0h there was a difference at the highest BSP1 concentration (40 μg/mL) (P <0.05) for ALH and BCF (control 2,53 ± 0,08a and 40 μg 2,40 ± 0,07b). After 1h TRT, the concentration of 40 μg / mL continued to present a significant difference. Hyperactivation is characterized by an increase in average lateral head displacement (ALH, mm), and beat cross frequency (BCF, Hz), possibly due to an increase in calcium influx. Our results indicate exactly the opposite with a significant reduction in these two parameters. These results suggest that in the horse BSP1 acts modulating calcium influx. Mitochondrial function determined by JC-1 showed a significant difference at the concentration of 10 μg/mL of the protein, lower than the other groups. As the bovine has a higher fertility compared to stallion, these results, in addition to the novelty of the work, allow further studies using proteins from heterologous species with the aim of improving seminal quality in stallions. In conclusion, BSP1 may exert an antioxidant effect in relation to the mitochondrial potential of sperm and change sperm kinematics due to a modulation of calcium influx by BSP1 protein. Further investigations are required to evaluate intracellular calcium concentrations after addition of BSP1.