Effects of green tea polyphenols and α-tocopherol on the quality of chilled cat spermatozoa and sperm IZUMO1 protein expression during long-term preservation

Sperm IZUMO1 protein was recently found to be a crucial mediator in the interaction and fusion with eggs, indicating an important role in assuring the favourable outcome from long-term preservation of chilled semen. The purpose of this study was to investigate whether supplementation of chilled semen extender with green tea polyphenols together with alpha-tocopherol would provide synergistic effects to prolong sperm survival and maintain IZUMO1 protein stability in cat spermatozoa. Sperm samples were collected from the cat epididymis before being diluted with semen extender containing various concentrations of alpha-tocopherol (0, 2.5, 5 and 7.5 mu g/ml) and 0.75 mg/ml green tea polyphenols and cooled to 4 degrees C. One sample without antioxidants served as a control. Sperm characteristics and IZUMO1 protein expression were investigated before and after chilling at 3, 6, 9, 12 and 15 days. Using alpha-tocopherol at 5 mu g/ml together with 0.75 mg/ml green tea polyphenols in the semen extender is the most suitable condition to retain the sperm characteristics up to nine days of preservation. Cat IZUMO1 proteins, 17 kDa, were identified at the equatorial segment of acrosome reacted sperm. Without antioxidant, cold storage can gradually degrade the IZUMO1 protein level. Sperm IZUMO1 protein was markedly conserved by supplementation of 5 mu g/ml alpha-tocopherol together with 0.75 mg/ml green tea polyphenols up to 12 days in cold storage. These findings indicate that green tea polyphenols and alpha-tocopherol have protective effects on the preservation of sperm characteristics and IZUMO1 protein integrity of cat epididymal sperm during long-term chilling.