Bovine Semen Preparation and Capacitation

Semen samples were obtained from 30 healthy adult Holstein Friesian bulls at a local breeding facility (Slovenské biologické služby, a.s., Nitra, Slovakia) with the help of an artificial vagina. All samples (1 sample per bull) were distributed into three fractions. The first and second ones were immediately transported into the laboratory and incubated for 30 min at 39 °C and 5% concentration of CO₂, either with physiological saline solution (IMUNA PHARM, A. S., Šarišské Michaľany, Slovakia) as a control or with a capacitation medium consisting of 100 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, USA), 3 mM potassium chloride (Sigma-Aldrich, St. Louis, MO, USA), 25 mM sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 283 µM sodium phosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 1.5 mM magnesium chloride (Sigma-Aldrich, St. Louis, MO, USA), 2.5 mM calcium chloride (Sigma-Aldrich, St. Louis, MO, USA), 0.37% sodium DL-lactate solution (60%; Sigma-Aldrich, St. Louis, MO, USA) and 0.2% phenol red solution (0.5%; Sigma-Aldrich, St. Louis, MO, USA) [84 (link)] diluted in a ratio of 1:40. The third part was cryopreserved for further investigation.