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

Abstract

Abstract. 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 4°C, 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.

Effect of epidermal growth factor on buffalo frozen spermatozoa biometry and metabolic activity

“…Former studies localized EGF receptors at higher extent in the acrosome region than to the post-acrosome and the flagellum [7]. Although no former studies verified the effect of EGF on frozen semen characteristics, fresh ram semen stored with EGF at 4 ℃ showed an enhanced motility, viability and membrane integrity [26]. EGF increased ram spermatozoa motility at all doses tested, especially when EGF was added to the spermatozoa after 2-day of storage.…”

Effect of caffeine on functions of cooling-stored ram sperm in vitro

“…Similarly, Maxwell et al (1995) reported no effect of caffeine on the acrosome integrity of frozenthawed ram spermatozoa. For successful fertilization, high incidence of sperm with intact membrane is necessary, because intact plasma membrane is an essential requirement for sperm cell function (Makarevich et al 2011). Spermatozoa with damaged or inactive membranes have limited viability and fertilization potential (Correa and Zavos 1994).…”

“…The sperm with PS translocation exhibited green fluorescence and was regarded as sperm with membrane destabilization. Presence of annexin V-positivity in sperm was localized on the acrosomal part of the sperm head, post-acrosomal segment, equatorial segment and on sperm membrane along the entire head (Makarevich et al 2011). …”

“…Staining solution contained 20 µmol·l Sperm membrane phosphatidylserine (PS) translocation (membrane destabilization) was detected using fluorescently labelled Annexin-V (Roche Slovakia Ltd, Bratislava, Slovak Republic). Sperm samples were prepared as described before by Makarevich et al (2011). The sperm with PS translocation exhibited green fluorescence and was regarded as sperm with membrane destabilization.…”

“…Sperm samples were not fixed, therefore, PNA marked only sperm with disturbed plasma membrane (green signal) whilst sperm with intact membranes remained unstained. Sperm samples were prepared as described before by Makarevich et al (2011). Staining solution contained 20 µmol·l Sperm membrane phosphatidylserine (PS) translocation (membrane destabilization) was detected using fluorescently labelled Annexin-V (Roche Slovakia Ltd, Bratislava, Slovak Republic).…”