In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

We studied resistance to manganese (Mn) toxicity under acidic conditions and its relationship with nutrients such as calcium (Ca) and magnesium (Mg) in new perennial ryegrass (Lolium perenne L.) genotypes (One-50, Banquet-II and Halo-AR1) introduced in southern Chile, using the Nui genotype as the reference. Plants were grown in nutrient solution at increased Mn concentrations (0-750 μM) at pH 4.8, and physiological and biochemical features were determined. Under higher Mn concentration, the One-50 genotype had a significantly lower relative growth rate (RGR) of shoots and roots, whereas in the other cultivars this parameter did not change under variable Mn treatments. Increasing the Mn concentration led to an increased Mn concentration in roots and shoots, with Banquet-II and Halo-AR1 having higher Mn in roots than shoots. Shoot Mg and Ca concentrations in all genotypes (except Banquet-II) decreased concomitantly with increasing Mn applications. In contrast to the other genotypes, Banquet-II and Halo-AR1 maintained their net CO assimilation rate regardless of Mn treatment, whereas the chlorophyll concentration decreased in all genotypes with the exception of Banquet-II. In addition, lipid peroxidation in Banquet-II roots increased at 150 μM Mn, but decreased at higher Mn concentrations. This decrease was associated with an increase in antioxidant capacity as well as total phenol concentration. Banquet-II and Halo-AR1 appear to be the most Mn-resistant genotypes based on RGR and CO assimilation rate. In addition, Mn excess provoked a strong decrease in Ca and Mg concentrations in shoots of the Mn-sensitive genotype, whereas only slight variations in the Mn-resistant genotype were noted. When other evaluated parameters were taken into account, we concluded that among the perennial ryegrass genotypes introduced recently into southern Chile Banquet-II appears to be the most Mn-resistant, followed by Halo-AR1, with One-50 being the most sensitive.

Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin-like, chymotrypsin-like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim-up method (4 x 10(6) cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N-alpha-p-tosyl-l-lysine-chloromethyl-ketone (trypsin inhibitor); Trypsin inhibitor (I-S Type from soybean); N-tosyl-l-phenylalanine-chloromethyl-ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm-zona binding capacity was evaluated using cumulus cell-free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome-reacted sperm was observed only in cells incubated with trypsin (10.2 +/- 1%) and chymotrypsin inhibitors (18.5 +/- 1%) in relation to the control (52.2 +/- 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 +/- 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.

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Acrosome reaction (AR) induced by low temperature has been used to evaluate sperm function; it correlates adequately with the fertilization percentages in vitro. In this study, the technique of AR induction by low temperature was used to evaluate the effect in the protection of the acrosome by cryopreservatives normally used in human semen cryopreservation. Donor sperm selected by use of the migration sedimentation technique was incubated in human tubal fluid medium, added to dimethyl sulphoxide 1 m, ethylene glycol 0.75 m, glycerol 1 m, incubated at 4 degrees C and 20 degrees C (as a control) for 18 h, and then for 3 h at 37 degrees C in a cell incubator. The AR was evaluated by triple stain in 100 viable spermatozoa. The effect of cryopreservatives on acrosome preservation in samples incubated for 18 h at 4 degrees C was as follows: 78% intact acrosome for glycerol, 77.8% intact acrosome for dimethyl sulphoxide and 96.2% intact acrosome for ethylene glycol (P < 0.0025 compared with glycerol and dimethyl-sulphoxide). The sperm samples incubated with cryopreservatives for 18 h at 20 degrees C did not show an increase in the percentage of AR in samples incubated with glycerol and ethylene glycol, while a significant variation was observed in the sample incubated with dimethyl sulphoxide (P < 0.001). Additional incubation for 3 h at 37 degrees C significantly increased the AR only in the sample incubated with glycerol (P < 0.001). Acrosome preservation is essential in the fertilization process and the evaluation of acrosome reaction induction by low temperature test was satisfactory. This test proves that ethylene glycol presents a greater protective effect on the acrosome preservation of human spermatozoa.