128 IMPROVED POST-THAW MOTILITY, VIABILITY, AND FERTILITY ARE ACHIEVED BY HYDROSTATIC PRESSURE-TREATED BULL SEMEN

Previously, we reported that high hydrostatic pressure (HHP) significantly improves post-thaw survival of frozen mouse and IVP bovine blastocysts, presumably from the induction of shock proteins (Pribenszky et al. 2005 Anim. Repr. Sci. 87, 143–150; 2005 Repr. Dom. Anim. 40, 338). We also have reported increased post-thaw survival of HHP-treated boar and bull semen (Pribenszky et al. 2004 Repr. Fert. Dev. 17, 199–200; 2005 Repr. Fert. Dev. 18, 162–163). We now report further data on the effect of HHP treatment on motility, viability, and fertility of frozen–thawed bull semen. HHP treatments were executed by a computer-controlled pressurizing device (Cryo-Innovation, Ltd., Budapest, Hungary). Semen of 21 bulls was diluted individually to a sperm concentration of 8 � 107 mL-1 with AndroMed� or Triladyl� extenders (MiniT�b, Tiefenbach, Germany). Diluted sperm was loaded into 0.25-mL straws at 25�C, and then divided into 2 groups: one group was treated with a HHP pulse defined and optimized earlier (30 MPa for 90 min); the other group was held at 5�C for the corresponding time. After HHP, treated samples also were placed at 5�C for 3–4 h. After equilibration at 5�C, samples were frozen (10 min at -110�C, and then plunged into LN2). Straws were thawed in a 35�C water bath for 30 s. Progressive motility was assessed by the CASA system (MiniT�b). Experiments were replicated twice for each bull. Paired t-test was used to analyze data. HHP treatment significantly increased the post-thaw motility of the frozen semen of the bull population examined. The mean of the differences was 21.14% (95% confidence interval: 13.56–28.72); P = 1.08 � 10-5. The post-thaw motility of 3, 7, and 5 of the 21 bulls increased by an additional 35–60%, 25–35%, and 10–25%, respectively; no effect was seen for 6 of the bulls. Four of the 7 bulls with low (3–19%) post-thaw motility were improved to the range of 43–70% by HHP treatment. Semen of 10 bulls was the subject of viability analysis individually. Sperm head, tail, and acrosome membrane integrity were evaluated with Kovacs-Foote staining (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) and counting 300 sperm/sample. Paired t-test was used to analyze data. With HHP treatment, the proportion of the cells with intact tail, head, and acrosome increased significantly (P d 0.008). Eighty-two cows were inseminated with HHP-treated frozen–thawed semen; the 60 days non-return rate was 90.24%, whereas the 60 days non-return rate in the same population and time without treatment was 82.3% (n = 4789). Data were analyzed with the exact binomial test. HHP treatment significantly improved the non-return rate (P = 0.035; 95% confidence interval: 0.83–1.00). HHP treatment substantially increases the post-thaw semen quality of the bull population. Also, the semen of a proportion of bulls with very low semen freezability can be increased to the range where it can be frozen commercially. Further investigations are needed, including large-scale field trials incorporating the insemination of the otherwise low freezers and the biological background of the process. This work was supported by GVOP-TST050157 and Besamungsanstalt Klessheim.