Addition of Serine protease inhibitors on equine sperm extender during cooling and freezing.

The objective of the present work was to examine the consequences of adding serine protease inhibitor and its synthetic analogs to stallion semen during cryopreservation in order to reduce cryocapacitation during cooling and freezing. Semen from six stallions was collected, and samples were cooled to 5oC following the addition of serine proteases inhibitors a) Benzamidine (5mM, 7.5mM, and 9mM); b) Berenil (1mM, 2.5mM, and 5mM); c) purified inhibitor (4mM) in final concentrations, and other samples were frozen with INRA 82 freezing extender in liquid nitrogen, following the addition of serine proteases inhibitors. Sperm progressive motility was evaluated under phase contrast microscopy, and membrane functional integrity with the hyposmotic swelling test (HOST). Sperm membrane integrity was assessed with propidium iodide stain and acrosome integrity (AI) with FITC-PNA stain, using a flow cytometer, and induction of acrosome reaction with calcium ionophore A23187 (5μM). In the control and treated groups, respectively, the mean values of progressive motility (47.5 % and 48.7 %) and membrane integrity (68.0% and 63.8%) after cooling and of progressive motility (10.8% and 11.8%), membrane integrity (21.4% and 23.4%), HOST+ (24.7% and 25.9%) and acrosome reaction (3.8% and 3.4%) after thawing, did not differ (p>0.05). After the induction of acrosome reaction, no difference (p>0.05) was observed in acrosome reacted sperm between the control (12.7%) and treated groups (16.8%). The tested serine protease inhibitors could not reduce stallion sperm membrane scrambling which occurs during cooling and freezing.