Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa

Although the effect of semen plasma on the function of spermatozoa has been widely studied, results are contradictory. We showed that semen plasma proteins are adsorbed onto the cold-shocked ram sperm surface, and that this adsorption is able to reverse the membrane alterations induced by cold-shock. In the present study we evaluate whether the addition of semen plasma proteins before the cold-shock would prevent membrane damage and maintain ram sperm viability. Ram spermatozoa freed from semen plasma by a dextran/swim-up procedure were strongly affected by the cold-shock treatment, lowering cell viability (membrane integrity by fluorescence markers) from 72.2+/-3.4% to 24.6+/-2.1%. Adding semen plasma proteins (> 3 kDa) to the medium before the cold treatment had an immediate beneficial effect on sperm survival in all samples. This effect was concentration-dependent, since the percentage of membrane-intact spermatozoa increased significantly with increased protein concentration in the incubation medium. The highest concentration of proteins (2.1 mg) continued to protect the membranes after 1 h of incubation at 20 degrees C while lower concentrations (0.7 and 1.4 mg) showed a slight decline. Inclusion of linoleic-oleic acids had a beneficial effect on preserving sperm viability when 25, 37 or 75 micromolar linoleic-oleic acids were added. There was a positive interaction between fatty acids and semen plasma proteins. Thus, the addition of 25 micromolar oleic-linoleic acid in the presence of 2.1 mg semen plasma proteins accounted for an increase in viability up to 50.7% significance (P<0.001) relative to the control sample (25%). Likewise, semen plasma proteins significantly promoted the ability of Vitamin E (alpha-tocopherol phosphate) to improve sperm survival. A 26% viability value obtained after cold-shock in the control sample significantly increased (P<0.001) up to 57% in the sample with 1.6 mM Vitamin E phosphate and 2.1 mg semen plasma proteins (0 h). This study demonstrates that impaired function of cold-shocked ram spermatozoa freed from semen plasma could be prevented by addition of semen plasma proteins, resulting in higher maintained viability values. Inclusion of either linoleic-oleic acids or vitamin E together with semen plasma proteins would increase the improvement in ram spermatozoa survival.