Efficacy of glutathione addition in egg yolk and soy lecithin based extenders of cryopreservation on bovine sperm viability

With the advances in animal reproduction biotechnology, the use of cryopreserved semen in artificial insemination stands out as an important tool for the genetic improvement of various animal species. However, the genetic impact on the artificial insemination industry is limited by the cryopreservation process, causing physical and chemical changes in the membranes of spermatozoa, leading also to the production of reactive oxygen species (ROS). In order to reduce the oxidative damage caused by high concentrations of ROS, antioxidants have been added in cryopreservation extenders of bovine semen. The present study aimed to evaluate the effect of glutathione addition in egg yolk (Bovimix®; Nutricell, Campinas, Brazil) and soy lecithin based extenders (AndroMed®; Minitube, Hauptstrasse, Germany) of cryopreservation, on post-thaw viability of bovine semen. Five Holstein bulls were used, being submitted to six semen collections. The ejaculates were split into two portions, diluted to a concentration of 40x10⁶ spermatozoa/mL, with egg yolk and soy lecithin-based extenders, Bovimix® and AndroMed®, respectively. Each portion was fractioned in three 15 mL centrifuge tubes, resulting in 6 treatments: 1. Control Bovimix® (BC), without adding antioxidants; 2. Bovimix® 1.5 mM glutathione (B1.5); 3. Bovimix® 2.5 mM glutathione (B2.5); 4. Control AndroMed® (AC), without adding antioxidants; 5. AndroMed® 1.5 mM glutathione (A1.5); 6. AndroMed® 2.5 mM glutathione (A2.5). After homogenization, the semen was packaged in 0.5 mL straws, arranged horizontally on a screened steel tray and maintained at 5° C for five hours. After the refrigeration period, the samples were kept for 20 minutes in nitrogen vapor, 3 cm above the nitrogen level. After frozen, they were stored in a nitrogen container. For analysis, the straws were thawed at 37 °C for 30 seconds and computer assisted sperm analysis (CASA) and analysis of sperm membrane integrity were evaluated. The percentage of sperm membrane integrity were evaluated by immunofluorescence microscopy with fluorescent probes (propidium iodide and carboxyfluorescein). Sperm variables assessed by CASA in the present study were total motility (MT-%), progressive motility (MP-%), curvilinear velocity (VCL-µm/s), straight line velocity (VSL-µm/s), average path velocity (VAP-µm/s), amplitude of lateral head displacement (ALH-µm), beat cross frequency (BCF-Hz), straightness (STR-%) and linearity (LIN-%). The data were analyzed using ANOVA and means comparison were analyzed by Tukey test at 5% of significance. There was no statistical difference in MT, MP, VCL, VSL, ALH, BCF and STR between treatments. However, the variable VAP was higher in all treatments in which the exterder AndroMed® were used (AC-68.42, A1.5-68.92, A2.5-65.44 x BC-56.10, B1.5-56.45, B2.5-56.57). The variable LIN was higher in all treatments in which Bovimix® was used (BC-51.00, B1.5-50.80, B2.5-49.73 x AC-42.96, A1.5- 44.43, A2.5-43.50). There was no statistical difference in the percentage of sperm membrane integrity between treatments. In conclusion, both treatments were efficient to maintaining post-thaw viability of semen. However, glutathione didn’t improve the sperm viability of the post-thaw bovine semen.