Functional characteristics of ram cooling-stored spermatozoa under the influence of epidermal growth factor

The aim of the study was to examine the effects of epidermal growth factor (EGF) on ram sperm traits following hypothermic storage. Fresh ram ejaculates were diluted in Triladyl extender, pooled and divided into groups according to EGF doses added (0, 100, 200 or 400 ng/ml). Following 72-96 h storage at 4ºC, the spermatozoa were stained for a plasma membrane integrity (PNA-FITC), membrane phosphatidylserine (PS) translocation (annexin V-Fluos) and apoptosis (Yo-Pro-1), and analyzed by fluorescent microscopy. Sperm motility was measured using computer-assisted sperm analysis (CASA) and sperm fertilizing ability was tested using in vitro penetration/fertilization test on bovine prematured oocytes. EGF increased sperm motility at all doses tested, decreased the proportion of spermatozoa with damaged plasma membrane (at 200 or 400 ng/ml), and decreased the proportion of apoptotic (Yo-Pro-1) spermatozoa when given at 200 or 400 (but not 100) ng/ml. The proportion of spermatozoa with PS translocations (8.5%) was affected by neither of the EGF concentrations tested. However, fertilizing ability of ram sperm in the in vitro test was not improved by EGF (200 ng/ml). In summary, EGF when given at higher concentrations improved sperm viability and motility after cooling storage, but these effects were not reflected in sperm fertilizing ability in vitro.