Abstract:
The process of cooling to 5�C prior to freezing produces physical and chemical stress on the sperm membrane associated with oxidative stress and reactive oxygen species (ROS) generation that reduces sperm viability and fertilizing ability. The addition of antioxidants to cooling medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of addition of reduced glutathione (GSH) to cooling extenders on (1) plasma membrane integrity, (2) acrosome reaction induction by ionophore A 23187 or progesterone, and (3) in vitro fertilization. Ejaculate-rich fractions from three mature pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15�C over 2�h (group C). Thereafter, sperm were centrifuged and diluted in lactose/egg-yolk extender with 0�mM (group 0), 1�mM (group 1) or 5�mM (group 5) of GSH, cooled to 5�C over 2�h. The acrosome reaction was then induced by 1�_M calcium ionophore or 10�_M progesterone in TALP medium and incubated in 5% CO2, 38.5�C for 30 or 45�min, respectively. Membrane integrity was evaluated by propidium iodide, and acrosomal status was monitored by means of FITC-labeled peanut agglutinin. Finally, in vitro fertilization was performed with these four spermatozoa groups as described previously (Mat�s et al. 2003 Reproduction 125, 133-141). ANOVA analysis revealed that the addition of GSH had no effect on the membrane integrity (ranged 58.8 to 66.9) or acrosome reaction induction (ranged 24.3 to 28.2, and 55.7 to 41.4 for progesterone and calcium ionophore, respectively). However, the results of the penetration assay revealed that the cooling affected the penetration rate and the number of sperm per oocyte (Table 1), and this assay is better than the others to predict changes in the spermatozoa functionality (Gadea J and Mat�s C 2000 Theriogenology 54, 1343-1357). In conclusion, the cooling process affects the in vitro fertilization, but the addition of GSH to the medium did not influence the parameters studied. Supported by AGL2000-0485-CO2-01.