Development of a sperm cryopreservation protocol for redside dace, Clinostomus elongatus: implications for genome resource banking

Populations of Redside Dace Clinostomus elongatus have declined in many areas across the species’North American range. Therefore, the development of sperm cryopreservation technology would provide an invaluable means of preserving genetic diversity in populations that are in imminent danger of extirpation.We developed cryopreservation protocols by testing the effects of diluent (buffered sperm motility-inhibiting saline solution [BSMIS]; BSMIS + glycine; sucrose; and Hanks’ balanced salt solution [HBSS]), cryoprotectant (dimethyl sulfoxide [DMSO]; propylene glycol [PG]; N,N-dimethylacetamide [DMA]; and methanol), freezing rate (1, 5, and 10◦C/min), and male-to-male variation on sperm quality.

Incubating sperm in extenders affected motility;BSMIS + glycine + methanol,BSMIS + glycine + PG, and HBSS + methanol were the only treatments for which motility was not significantly different from that of fresh sperm. Sperm frozen with sucrose had higher motility than sperm frozen with BSMIS + glycine, and sperm frozen with DMSO had higher motility than sperm frozen with methanol.

Freezing rates were evaluated for BSMIS + glycine, HBSS, and sucrose; all diluents were frozen with DMSO. The effect of freezing rate was not significant for BSMIS + glycine or for HBSS, but an effect was detected for sucrose, with sperm frozen at 5◦C/min or 10◦C/min having higher motility than sperm frozen at 1◦C/min.

The effect of extender was not significant at 1◦C/min or 5◦C/min, but an effect was detected at 10◦C/min such that sperm frozen with sucrose had the highest motility.Male-to-male variability was evaluated by using sucrose + DMSO and a freezing rate of 10◦C/min. For these males, the sperm motility recovery index ranged from 6.67% to 79.27%, and the sperm velocity recovery index ranged from 21.37% to 57.33%.

Our findings demonstrate that cryopreservation of Redside Dace sperm in a sucrose + DMSO extender at a freezing rate of 10◦C/min is adequate for preserving genetic diversity via sperm banks