Sperm washing method alters the ability of seminal plasma proteins to revert the cold‐shock damage on ram sperm membrane

Abstract

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim‐up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold‐shocked sperm. Seminal plasma proteins were added to cold‐shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter‐current distribution (CCCD) in an aqueous two‐phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold‐shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane‐intact spermatozoa, i.e. propidium iodide‐negative. The addition of 700 μg of SPP to the swim‐up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% ( p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold‐shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim‐up. However, this restorative effect was again much lower when SPP were added to the cold‐shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold‐shocked sperm. Separation of spermatozoa by swim‐up accounts for higher viability recovery than that obtained by filtration washing.