Uso da Binder of Sperm Protein 1 (BSP1) bovino na congelabilidade de sêmen de garanhão

Abstract

It is reported a beneficial effect of seminal plasma proteins from bulls in cryopreservation of several species. Some seminal plasma proteins has a positive effect on sperm cell, including sperm capacitation, interaction with oviductal epithelium with a positive effect on fertilization. This work aimed to purify the Binder of SPerm 1 (BSP1), protein present in the seminal fluid of bulls by ammonium sulfate precipitation and observe the effect of addition of this protein in stallion semen congelability. In experiment 1, BSP1 purification was performed by precipitation with ammonium sulfate technique. Seminal fluid was extracted from vesicular glands obtained from ten bulls in a commercial slaughterhouse. After extraction, a protein inhibitor was used and a pool of all samples were stored at -20ºC, separating a portion to determine concentration. To check process of purification, SDS-PAGE and Western blot was performed confirming presence of BSP1. Protein presence was observed in all the fractions on average of 98%. With this protein, experiment 2 was conducted to evaluate the effect of addition of BSP1 to stallion semen at concentrations of 0 (control), 10, 20 and 40 μg/mL to a commercial extender (BOTUCRIO®), in the 4 quarter horse stallions, in a total of 5 ejaculate by sire. After evaluation, semen was centrifuged, diluted to freeze adding BSP1 protein, in a freezing programmable machine (TK 3000®). Significant difference was seen in sperm kinetic parameters (ALH, BCF, WOB) both in “0” time and “1 hour” incubation time as well (P < 0.05). There was also a difference in mitochondrial membrane potential, when added the protein at a concentration of 10 μg/mL, being lower than the other concentrations and control. The addition of the BSP1 did not cause any damage in acrosomal or plasma membrane integrity, even being from heterologous species. As the bull presentes a high fertility compared to stallion, this study provides a good perspective on the use of heterologous proteins in order to improve seminal quality in stallions.