246 EFFECT OF NITRIC OXIDE INHIBITION (N-ω-NITRO-L-ARGININE METHYL ESTER) AND SCAVENGER (METHYLENE BLUE) ON PLASMA MEMBRANE PEROXIDATION OF EQUINE CRYOPRESERVED SPERM
Authors: Silva D. F. , Andrade A. F. C. , Caldas-Bussiere M. C. , Celeghini E. C. C. , Alonso M. A. , Carvalho H. F. , Lemes K. M. , Florez-Rodriguez S. A. , Affonso F. J. , Arruda R. P. 
Published: December 2012

The peroxidation of plasma membrane lipids has been claimed to be a major factor involved in sublethal cryodamage. Some authors have reported that reactive oxygen species (ROS) accumulation could be an important factor leading to further damage in post-thaw sperm. Oxidative stress occurring in the sperm is a phenomenon associated with increased rate of oxidation of cellular components and excessive production of ROS and nitrogen. The objective of this study was to evaluate the effect of NO inhibition [N-ω-nitro-l-arginine methyl ester (l-NAME)] and scavenger (methylene blue) on the plasma membrane peroxidation of equine cryopreserved capacitated sperm. Three ejaculates were obtained from each of three stallions (n = 9). Semen was packaged into 0.5-mL straws to a concentration of 200 × 106 sperm mL–1 in BotuCrio® extender and frozen using an automated technique with a programmed machine. Four straws were thawed in a water bath at 37°C for 30 s and centrifuged in bovine IVF media supplemented with sodium bicarbonate and BSA for the capacitation of equine sperm. The supernatant was withdrawn, and semen was then incubated in the same media with l-arginine, with or without the NO synthase inhibitor, l-NAME, and in media with l-arginine with or without the NO scavenger, methylene blue: treatment (T)1 = control; T2 = 10 mM l-arginine (based on previous experiments); T3 = 1 mM l-NAME; T4 = 100 mM methylene blue; T5 = 10 mM l-arginine + 1 mM l-NAME; and T6 = 10 mM l-arginine + 100 mM methylene blue for 60, 120, and 300 min at 38°C under 5% CO2. After incubation, cells presenting membrane peroxidation were identified using an association of the fluorescent probes C11-BODIPY581/591 (1 mg mL–1; D-3861) and PI (0.5 mg mL–1; L0770). Nitric oxide production was identified using a 4,5-diaminofluorescein-2/diacetate (10 µM) probe associated with PI. The H33342 probe was used to avoid those particles presenting the same size and granularity as sperm cells being included in the counting. Both evaluations were performed using flow cytometry. Data were analysed by ANOVA, and the means were compared within each time with Tukey’s test, with a level of significance of 5%, using SAS software (SAS Institute Inc., Cary, NC, USA). Lipid peroxidation of the membrane was not influenced by the treatments at the different times (P > 0.05). This characteristic was reduced for the groups treated with methylene blue (T4 and T6) at times 60, 120, and 300 min compared with the other treated groups. Nitric oxide produced by sperm was not influenced by treatment at different times (P > 0.05). However, methylene blue addition was able to decrease NO production in treatments T4 and T6 at all the incubation times evaluated compared with the other treatments. Therefore, methylene blue removed NO and decreased plasma membrane lipid peroxidation, suggesting an antioxidant role of this scavenger. On the other hand, more research is needed to elucidate the mechanism of action of methylene blue on the physiology of cryopreserved equine sperm.
Supported by FAPESP (grant 2009/54906-5).