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Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.