Isoglycyrrhizin supplementation of frozen goat semen-extender improves post-thaw sperm quality and in vitro fertilization rates

This study examined the effect of supplementation of freezing extender with isoglycyrrhizin (ISL), a natural antioxidant agent, on the quality and fertility potential of goat spermatozoa after cryopreservation. Forty ejaculates were collected from eight Chongming White rams and diluted with five concentrations of ISL: 0 (control group), 50, 100, 150, and 200 μg/mL. The quality, motility parameters, antioxidant properties, mRNA expression of antioxidant and ferroptosis genes, and ability to induce fertilization, were evaluated following freezing/thawing. Total motility and progressive motility were significantly increased in spermatozoa following the addition of 150 and 200 μg/mL ISL. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities were enhanced in all ISL-supplemented groups compared to controls. Adding ISL decreased the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO). mRNA levels of ferroptosis-related genes, voltage-dependent anion channel protein 2 (VDAC2), voltage-dependent anion channel protein 3 (VDAC3), protein 53 (P53), and nuclear factor erythroid 2-related factor 2 (NRF2) tended to decrease after adding ISL, whereas the levels of antioxidant genes glutathione peroxidase 4 (GPX4) and glutathione peroxidase 4 (GPX5), tended to increase. The best spermatozoa quality and the strongest antioxidant properties were obtained after adding 150 μg/mL ISL. Therefore, fresh semen, frozen semen, and frozen semen with 150 μg/mL ISL was used for in vitro fertilization of oocytes. The cleavage and blastocyst rates were significantly lower in frozen semen compared with fresh semen, whereas frozen semen containing 150 μg/mL ISL showed a significant increase in cleavage and blastocyst rates compared with frozen semen. In conclusion, ISL can be used as an antioxidant in goat semen cryodilution, and the addition of ISL can improve the quality and antioxidant properties of frozen and thawed spermatozoa. ISL can also protect the ability of spermatozoa to be fertilized and develop; 150 μg/mL ISL was the optimal concentration for addition.