title: Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa.
abstract: 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 microM linoleic-oleic acids were added. There was a positive interaction between fatty acids and semen plasma proteins. Thus, the addition of 25 microM 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.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Zaragoza, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Pérez-Pé
authors.forename: J A
authors.initials: JA
authors.lastname: Cebrián-Pérez
authors.forename: T
authors.initials: T
authors.lastname: Muiño-Blanco
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D003080:Cold Temperature / Q000009:adverse effects*; D004396:Coloring Agents / Q000737:chemistry; D019787:Linoleic Acid / Q000494:pharmacology / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000502:physiology*; D008858:Microscopy, Phase-Contrast / Q000662:veterinary; D019301:Oleic Acid / Q000494:pharmacology / Q000502:physiology; D011419:Propidium / Q000737:chemistry; D012661:Semen / Q000187:drug effects / Q000502:physiology*; D012756:Sheep / Q000502:physiology*; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D014810:Vitamin E / Q000494:pharmacology / Q000502:physiology
chemical_list: D004396:Coloring Agents; D008565:Membrane Proteins; D014810:Vitamin E; D019301:Oleic Acid; D011419:Propidium; D019787:Linoleic Acid