Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5 ℃

Abstract

The objective of this study was to evaluate the effects of reduced glutathione (GSH) supplementation on sperm motility, kinematics, mitochondrial activity and the abundance of sperm hexose transporters during liquid storage of ram semen at 5 ℃. Semen samples from eight rams were pooled and diluted with Tris-egg yolk extender supplemented with 0 (control), 50, 100 and 200 mM GSH, and they were assessed at different processing periods (fresh diluted, stored for 24, 48 and 72 h at 5 ℃). Sperm motility and kinematic parameters were analyzed by computer-assisted sperm analysis (CASA). The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of sperm membrane. Eosin-nigrosin and JC-1/PI staining methods were used to assess sperm viability and mitochondrial activity, respectively. The antioxidant status of sperm was examined using biological assays. The changes of sperm hexose transporters (GLUT 3, 5 and 8) were determined using western blot analysis. As compared to the control group, supplementation with 100 and 200 mM GSH improved (P ＜ 0.05) sperm total motility, membrane integrity, motion parameters and mitochondrial activity at 72 h of storage, as well as resulted in the higher sperm antioxidant status after storage from 24 to 72 h. The cooling induced a reduction of GLUT 3 and 8, and GSH treatment at 200 mM retarded the gradual decline of these proteins during the prolonged storage time. Our data suggest that GSH can reduce the decline of semen quality by influencing the antioxidant status and energy metabolism of liquid stored ram spermatozoa. The concentration of 200 mM could be appropriate for ram semen during liquid storage at 5 ℃.