Formerly we reported that a sublethal shock, high hydrostatic pressure (HHP), significantly improves the post-thaw survival of frozen mouse blastocysts and post-thaw motility of frozen bull sperm, presumably from the HHP induced changes in the protein profile [e.g. heat shock proteins (HSPs)] (Pribenszky et al. 2005 Rep. Fert. Dev. 17, 199–200; Pribenszky et al. 2005 Anim. Rep. Sci. 87, 143–150). We now report the effect of HHP on the motility of fresh boar semen, and we compare post-thaw motility of HHP-treated frozen boar semen with non-pressurized frozen-thawed controls. Pressurization was done both at RT and at body temperature (BT). Exp. 1: Semen was extended with Beltsville thawing solution (BTS), centrifuged, diluted with lactose-egg yolk diluent with a final concentration of 6% glycerol and 0.5% Equex (Minitüb, Tiefenbach, Germany) to a sperm concentration of 3 × 109/mL. Diluted sperm were loaded into 0.25-mL straws (IMV, Paillette Crista, France) at RT. Straws were heat sealed then assorted to one of the treatment groups (100, 200, 400, or 800 bar for 40, 80, or 120 min). Non-pressurized samples were left at RT for the corresponding time. For freezing, straws were put at 15°C for 3 h, and then 5°C for 2 h, followed by 3 cm above LN2 for 10 min before plunging into LN2. Exp. 1 was repeated on two boars. Exp. 2: Sperm was treated with 400 bar for 80 min at RT or BT, and was then prepared and frozen as described above, together with non-pressurized controls. Exp. 2 was repeated in five boars. For evaluation, straws were thawed in a 37°C water bath for 2 min. Motility was analyzed with CASA SpermVision 3.0 (Minitüb). For hypothesis testing, a linear mixed model was fit to the motility data. The analysis was carried out in R statistical software. In Exp. 1, the 100-, 200-, and 400-bar treatments did not affect motility; 800 bar treatments resulted in reduced motility compared to control. After 5 h of cold acclimatization, only the groups with 800 bar treatments and the non-pressurized controls had significantly reduced motility. After freezing-thawing motility (% ± SE) in groups pre-treated for 80 min with 200 or 400 bar was 43.2 ± 5.24 and 42 ± 3.24, respectively; control: 23.2 ± 1.83 motility in groups pre-treated for 120 min with 200 or 400 bar was 51 ± 2.33; 55.5 ± 3.63, respectively; control: 41.88 ± 2.97. The pre-treated groups displayed significantly enhanced motility compared to the nontreated controls. In Exp. 2, the HHP treatment performed at BT yielded the highest post-thaw motility compared to the HHP treatment at RT, or the non-pressurized controls (59.75 ± 2.59; 46.43 ± 2.05; 37.37 ± 2.19, respectively). All of these results differed significantly from each other. In conclusion, HHP treatment, simply inserted before the freezing step, can significantly increase post-thaw motility and yield consistent acceptable results. The effect of the treatment is even stronger at BT, but great care has to be taken to maintain BT from the time of sperm collection till the end of the treatment. Further experiments are being conducted concerning the pressure-induced alterations in the protein profile of boar spermatozoa (fresh and frozen-thawed).
This work was supported by OTKA061975 and TST050157.
Development of new methods for increasing the germination of licorice (Glycyrrhiza uralensis Fisch.) seeds: preliminary data
