Effects of L-Glutathione and Alpha-Tocopherol Added to Extenders on Sperm Quality in Cryopreservation of Epididymal Rat Spermatozoa

Ordek R., Yildiz C.

JOURNAL OF THE HELLENIC VETERINARY MEDICAL SOCIETY, vol.76, no.3, pp.9819-9828, 2025 (SCI-Expanded, Scopus)

Abstract

Freezing-thawing processes are believed to be a cause of stress associated with increased reactive oxygen species (ROS) generation, causing cell damage owing to oxidative stress. Glutathione is one of the important endogenous antioxidants present in all mammalian tissues. Alpha-tocopherol is a natural non-enzymatic antioxidant that protects sperm cells from ROS damage and prevents lipid peroxidation. In this study, the effects of L-glutathione (2.5, 5, and 10 mM) and alpha-tocopherol (0.1, 0.2, and 0.5 mM) at different doses added to 8% lactose + 23% egg yolk + 10% tris solution sperm extender on the freezing of rat spermatozoa liquid nitrogen were investigated as antioxidants. Spermatological analyses of the samples were performed after the frozen straw were thawed in a 37 degrees C water bath for 30 seconds. Compared with the control group after freezing and thawing, the highest motility was obtained in the 0.1 mM alpha-tocopherol (23.89 +/- 1.82%) and 2.5 mM L-glutathione (28.00 +/- 2.71%) groups (p<0.01). No significant difference was found between groups in terms of the rate of dead spermatozoa and abnormal acrosomes (p>0.01). Compared with the control group, the highest plasma membrane integrity was found in the 0.1 mM alpha-tocopherol and 2.5 mM L-glutathione groups (p<0.01). The highest motility values for spermatozoa endurance were determined in the 0.1 mM alpha-tocopherol and 2.5 mM L-glutathione groups compared to the control group (p<0.01). The rate of apoptotic and necrotic cells was lowest in the 0.1 mM alpha-tocopherol and 2.5 mM L-glutathione groups compared to the control group (p<0.01). As a result, it was determined that the addition of 0.1 mM alpha-tocopherol or 2.5 mM L-glutathione to the spermatozoa extender increased the sperm motility, functional plasma membrane integrity and motility endurance and decreased the evaluation apoptosis after freezing-thawing, and thus could be used successfully in the freezing extender of rat spermatozoa.