98 THE EFFECT OF HIGH HYDROSTATIC PRESSURE ON THE MOTILITY OF FRESH AND FROZEN-THAWED BULL SEMEN

Previously, we reported that a sublethal shock, high hydrostatic pressure (HHP), significantly improves the post-thaw survival of frozen mouse blastocysts, presumably from the induction of shock proteins (Pribenszky et al. 2004 Reprod. Fert. Dev. 16, 181). Others reported that HSP90 in spermatozoa decreased substantially after freezing (Huang et al. Theriogenology 51, 1007–1016; Cao Wen-Lei et al. 2003 Asian J. Androl. 5, 43–46). We now report the effect of HHP on motility of the fresh bull semen to determine whether sperm survives in an altered pressure environment, and to compare post-thaw motility of HHP-treated frozen bull semen with controls. The survival rates were compared by chi-square test. Expt 1: Semen of one bull was diluted to a sperm concentration of 8 × 107/mL with AndroMed extender (MiniTüb, Tiefenbach, Germany). Diluted sperm was loaded into 0.25-mL straws at 25°C. Each straw was cut in half. One demi-straw was heat-sealed and exposed to HHP, and sperm in the companion demi-straw served as a control. Experiments were replicated eight times for each pressure/time treatment. Progressive motility was assessed independently by light microscopic investigation by two individuals. The treatment groups were: 10 MPa for 30, 60, 90, or 120 min; 30 MPa for 30, 60, 90, 120, or 510 min; 50 MPa for 30, 60, or 90 min; 70 MPa for 30, 60, or 90 min; and 90 MPa for 30, 60, 90, 120, or 510 min. The average motility of the control samples ranged from 75 to 90%, while the average motility of the pressurized samples ranged between 55 (90 MPa/120 min) to 84% (10 MPa/30 min). The groups of 30 MPa/510 min and 90 MPa/510 min exhibited significantly lower motility compared to the other pressurized groups (27% and 33%, respectively; P < 0.05). Expt 2: Semen was collected from two bulls with poor sperm freezability. Semen was diluted as described for the first experiment, loaded into straws, and assigned to one of 4 treatment groups. Half the straws from each bull were exposed to 90 MPa/30 min, 90 MPa/90 min, 30 MPa/30 min, or 30 MPa/90 min, and then cryopreserved. Controls consisted of straws that were cryopreserved without pressure treatment. Cryopreservation steps were 60 min equilibration at 5°C, followed by 10 min at −110°C, and then plunging into liquid nitrogen. Straws were thawed in a 35°C water-bath for 30 s. Each treatment and control group was replicated 8 times (8 samples per bull). The average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized groups compared to the samples frozen without previous pressurization (P < 0.001) (Bull I: 2–3% without pressurization vs. 17–33% with pressurization; Bull II: 0% without pressurization vs. 21–35% with pressure pre-treatment). Among the pressure/time parameters used, 30 MPa/90 min proved significantly superior (33 and 35%; P < 0.05) for each of the bulls. Expt. 2 clearly demonstrates the beneficial effect of a previous pressure treatment on post-thaw motility of bull semen cryopreserved in our experiment. Further investigations are needed, including samples from different bulls, different freezing protocols, and the biological background of the process. This work was supported partly by NKFP 4/040/2001.