Standardization of fertilization protocols for sterlet Acipenser ruthenus is crucial for improving reproductive techniques and for conservation purposes. Our objectives were to determine the number of sperm (tested 430,000:1, 43,000:1, 4300:1, 430:1 sperm to egg) required to fertilize eggs and explore how pre-incubation of eggs in freshwater for 0min, 0.5min, 1min, and 10min interacts with different sperm ratios. Fertilization success ranged from 29.7% at 430:1 to 84.2% at 430,000:1. Pre-incubation time had no effect on fertilization success at 430,000:1 and 43,000:1 sperm to egg ratios, while it was significant at the 4300:1 and 430:1 ratios. The use of adequate experimental suboptimal sperm to egg ratio revealed a positive effect of pre-incubation time, such that at the 430:1 ratio, 0.5min pre-incubation increased the fertilization rate than 10min. At 0min pre-incubation the proportion of fertilized eggs increased at the 430,000:1 ratio, while at 1min fertilization increased at the 4300:1 ratio. At the 10min pre-incubation time, fertilization increased at the 43,000:1 ratio. Moreover, at the 0.5min pre-incubation time, the 43,000:1 ratio increased the fertilization rate than the 430:1 ratio. Generally, for 430:1 ratio, the fertilization rate is lower than in control. Transmission electron microscopy showed that pre-incubation of eggs in water for <10min does not trigger a cortical reaction or the formation of a perivitelline space. Results suggest that with a low sperm to egg ratio 0.5 to 1min pre-incubation of eggs in freshwater prior to fertilization can enhance fertilization rate of sterlet.