Ações da binder of Sperm protein 1 (BSP1) sobre a funcionalidade de espermatozoides bovinos: reação acrossômica, taxa de clivagem e produção in vitro de blastocistos

Abstract

The Binder of Sperm Protein 1 (BSP1) is a protein produced by seminal vesicles of animals. It helps into the fertilization process of oocytes, promoting sperm capacitation, mediating the ligation from itself with the oviduct epithelium, and increasing the motility in the oviduct. When spermatozoa with purified BSP1 were incubated, acrosomal reaction rates were determined with and without the presence of heparin, as controls. The percentage of acrosomal reaction was higher (p <0.05) in control 1 (44.5%) compared with the other treatments (BSP1:10 µg/mL: 24.5%; 20 µg/mL: 25.5%; 40 µg/mL: 26.0 %), and lower the rate (p> 0.05) when sperm were incubated only in Fert-TALP medium without heparin (14.5%). Cleavage and blastocyst rates were evaluated on days two and seven respectively, after fertilize oocytes in a medium containing BSP1. 1274 cumulus- oocyte complexes were recovered and incubated for 18 hours with frozen-thawed ejaculated semen in a Fert-TALP medium, containing: only heparin, 10, 20 or 40 μg/mL of BSP1. Cleavage and blastocyst rates (34.1 ± 4.4 vs 40.8 ± 5.0%) were similar (p> 0.05) when incubation was made with 10 µg/mL of BSP1 and heparin, respectively. However, the concentrations of 20 and 40 µg/mL decreased the formation of blastocysts (22.4 ± 2.9 and 19.3 ± 4.1%) compared with heparin (40.8 ± 5.1%, p <0.02). Subsequently, cumulus- oocyte complexes (n = 2239) were incubated with frozen-thawed ejaculated semen. Spermatozoa were selected by a gradient of 45% -90% Percoll® containing: only heparin, 10, 20, 40 μg/mL of BSP1 and there was a control 2 of Percoll® without heparin or BSP1. Cleavage rate was similar (p> 0.05) for the incubation with concentrations of 10, 20 and 40 μg/mL of BPS1 (67.7 ± 3.0, 68.7 ± 3.5, 75.2 ± 3.8, respectively). However, these rates were similar when it was used just heparin and when oocytes were incubated with 40µg/mL of BSP1 (78.9 ± 1.7 vs 75.2 ± 3.8%). For blastocyst rates was found that they were similar (p> 0.05) when the selection was made with heparin (44.1 ± 4.3%) and 40 µg/mL of BSP1 (30.0 ± 3.3%). Finally, 1213 cumulus-oocyte complexes were recovered and incubated with freeze-thawed epididymal semen in Fert-TALP medium, which contained: only heparin, with heparin, and 10, 20 or 40 µg/mL of BSP1. Cleavage and blastocyst rates were similar after incubation with and without heparin, but the concentration of 40µg/mL of BSP1 produced higher cleavages rates (79.0 ± 1.1%) than control 1 (68.5 ± 1.3%, p <0.05). About blastocyst rates, there were rates significantly higher when concentrations of 20 and 40 µg/mL of BSP1 were used (35.6 ± 2.5 and 41.1 ± 2.0%) than with (24.7 ± 3.2; p <0.05) and without heparin (27.3 ± 1.6%; p <0.0003). In conclusion, BSP1 allowed proper cleavage and development, when the protein was added to the fertilization medium and the Percoll® gradient using ejaculated semen, and to the fertilization medium when used epididymal sperm, without requiring the use of heparin.