371 DOES Percoll™ GRADIENT CENTRIFUGATION CAUSE HYPERACTIVATION ON CRYOPRESERVED BOVINE SPERMATOZOA?

Abstract

The density difference of 0.06% between X- and Y-bearing bovine spermatozoa has the potential to provide for separation X-bearing spermatozoa on specific gradient solutions of Percoll™ (Hossepian de Lima VFM et al. PCT/BR2004/000009). The question remains whether Percoll™ density gradient centrifugation induces the hyperactivation of spermatozoa, because it is believed that some decapacitating proteins are removed from the sperm surface during the process. Thus, the objective of this study was to evaluate if Percoll™ centrifugation causes hyperactivation of cryopreserved bovine spermatozoa. Semen doses were collected from six bulls of different breeds, including three taurine and three Zebu animals, and four ejaculates per bull were evaluated. The semen samples were thawed and evaluated before (control) and after (sexed group) centrifugation (500 g) in discontinuous Percoll™ density gradient (Hossepian Lima VFM et al. PCT/BR2004/000009). Sperm motility was assessed by Computer-Assisted Semen Analysis (CASA, Hamilton Thorne Biosciences, Beverly, MA, USA) and the results (mean ± SEM) were submitted to ANOVA. Higher (P < 0.0001) total and progressive sperm motilities were observed in sexed (85.0 ± 1.8 and 75.9 ± 1.7%, respectively) than in the control group (69.9 ± 1.7 and 59.2 ± 1.6%, respectively). The average path velocity, straight-line velocity and curvilinear velocity (VCL) were lower (P < 0.01) in sexed (91.3 ± 1.1, 79.8 ± 0.8, and 137.6 ± 3.5 (im/s, respectively) than in control group (107.6 ± 4.8, 91.1 ± 4.1, and 173.2 ± 8.9 (im/s, respectively). The amplitude of lateral head displacement (ALH) was higher (P < 0.0001) in control (6.7 ± 0.3 μm) than in sexed semen (5.1 ± 0.2 μm) and beat cross frequency was higher (P < 0.05) in sexed (35.9 ± 0.9 Hz) than in control semen (33.1 ± 1.0 Hz). The straightness and linearity (LIN) were higher (P < 0.01) in sexed (87.4 ± 0.7 and 61.4 ± 1.4%, respectively) than in control semen (84.4 ± 0.7 and 55.5 ± 1.2%, respectively). Regarding the results obtained in the present study, it was not possible to infer that the method used induced sperm hyperactivation. According to Marquez and Suarez (2007 Biol. Reprod., 76, 660-665), an increase in pH and intraflagellar calcium plays a key role in the axoneme changes, promoting an increase in ALH (from 9 to 13 μm) and VCL (from 200 to 315 μm/s), as well as a decrease in LIN (from 57 to 25%), which characterizes the presence of hyperactivated cells in the sample. In the present study, centrifuged spermatozoa presented significantly lower ALH and VCL and higher LIN compared to control semen samples. Therefore, the presence of glucose in the Percoll™ stock solution and constant pH monitoring could have prevented alkalinization of the medium, thereby preventing the occurrence of intraflagelar calcium influx and the consequent triggering of in vitro sperm hyperactivation.