Peroxidação lipídica da membrana plasmática de espermatozoides caprinos e ovinos submetidos à criopreservação com Trolox

Abstract

With the aim of identify the best method to assess lipid peroxidation in goat and sheep sperm, semen samples were diluted in skimmed milk (Glycerol 7%) and Tris-egg yolk (Glycerol 5%) extenders, respectively, and frozen (-196 °C). After thawing (37°C/30s), samples were analyzed to lipid peroxidation by spectrophotometry and high performance liquid chromatography – DAD (TBARS method) and flow cytometry (C11-BODIPY581/591). It was observed that peroxidation levels obtained by spectrophotometry were significantly higher than those found by HPLC method. C11-BODIPY581/591 complemented the data obtained by TBARS (HLPC) method, helping to better understand the results and providing an overview of damaged caused to cells. In conclusion, the dosage method of TBARS by HPLC and C11-BODIPY581 are more appropriated to assess lipid peroxidation in goat and sheep sperm, and it can be used with success together or separated,. After determine the best method to assess plasma membrane lipid peroxidation of goat and sheep sperm cryopreserved with Trolox, semen samples were diluted in skimmed milk (goat) or tris-egg yolk (sheep) extenders, without antioxidants (control group) or with Trolox at 20 μM or 40 μM/mL. After thawing at 37oC for 30 seconds, samples were submitted to assessment of lipid peroxidation by high performance liquid chromatography (HPLC) and flow cytometry (C11-BODIPY581/591), as well as plasma membrane integrity, acrosomal integrity and sperm kinematics. Semen extenders and fresh semen samples were also assessed by HPLC. No significant difference (P>0.05) was observed among experimental groups of both species to sperm kinematics, plasma membrane and acrosomal integrity. Significant difference also was not observed (P>0.05) among treatment groups to lipid peroxidation reductionIn conclusion, the cryopreservation process did not trigger lipid peroxidation on goat and sheep sperm plasma membrane, independent of Trolox addiction (20 e 40 μM).