Protein and lipid species in seminal plasma of fertile Holstein-Friesian bulls

Protein and lipid molecules in seminal plasma (SP) collected from fertile bulls were investigated. Semen was collected from 10 bulls (2 ejaculates each) and examined for standard semen analysis. Raw SP was recovered by centrifugation and total protein (TP) concentration was determined using a refractometer. Raw SP was desalted using a Sephadex G-25 desalting column then both raw and de-salted SP was subjected to SDS-PAGE. Neutral lipids and phospholipids of raw and desalted SP were separated by thin-layer chromatography (TLC). The results revealed that, all bulls had normal semen characteristics and TP concentration in SP ranged from 7.0 to 10.4 g/dL except bull No. 6 had a relatively low concentration of 4.9 to 6.8 g/dL. Neither proteins nor lipids species were different between raw and desalted SP. Seventeen proteins were detected ranging from 8.5 to 185.8 kDa, and those of 12, 13.5, 15, 21, 23 and 38 kDa were predominant. Notably, proteins of 10, 17.5, 19, 21, 80 and 185.8 kDa might be new candidates of SP proteins (SPPs). The detected neutral lipid spots corre-sponded to cholesterol, 1,2-dimyristoyl glycerol, 1,2-dioleoyloglycerol, 1,3-dimyristoyl glycerol and 1,3-dioleoyloglycerol. The detected phospholipids spots corresponded to non-migrating phospholipids, sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), cerebroside and polyglycerol phosphatide. Cholesterol represents the major molecule of neutral lipids, whereas SM, PC, PI and PE represent the major phospholipids. Noteworthy, there were 2 species of diacylglycerol (DAG) and 3 species of PI in bovine SP. In conclusion, this study gave a general picture of SP protein and lipid species in fertile bull semen, which might serve as fundamental knowledge for either semen analysis or prediction of male fertility

Desalted and lyophilized seminal plasma increases protein tyrosine-phosphorylation of frozen-thawed bull spermatozoa incubated with a cell-permeable cyclic AMP (cAMP) analog (cBiMPS)

The present study investigates the effect of desalted seminal plasma (SP) added to semen extender on hyperactivated motility and protein tyrosine-phosphorylation (PTP) of bull spermatozoa. The SP was harvested by centrifugation and desalted using Sephadex G-25 columns in order to be added to semen extender at 0 (control), 2.5, 12.5 and 25 mg/ml. Frozen-thawed spermatozoa were incubated with a cellpermeable cyclic AMP (cAMP) analog (cBiMPS) and examined subjectively for hyperactivated motility and for PTP by Western blotting. Although, the added SP sustains sperm motility at all incubation times especially in the presence of cBiMPS but without significant difference from the control samples. Moreover, total sperm motility of 12.5 and 25 mg/ml in the presence of cBiMPS at 60, 120 and 180 min were similar (P ≥ 0.05). Surprisingly, cBiMPS-incubated spermatozoa in the presence of desalted SP were capable of exhibiting hyperactivated motility. Addition of SP increased and prolonged intracellular cAMP-induced PTP and in total 21 phosphorylated proteins with molecular weight ranging from 10 to ＞230 kDa were detected. The most prominent tyrosine-phosphorylated proteins (TPPs) were of 32, 38, 74 and 80 kDa which were more predominant in fertile bulls than subfertile bull. Furthermore, TPPs of 45 and 48 kDa were cBiMPS-dependent in fertile bulls whereas, in subfertile bull the latter was barely detectable and the former was cBiMPS-independent at only 0 min. This increase in PTP not only emphasizing the beneficial roles of desalted SP but excluding any detrimental effect of it on sperm cell functions during storage as well