摘要：AbstractDensity gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of theRhamdia qhelenand to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm ofR. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.Graphical abstractGraphical abstract of the standardization of the density gradient centrifugation method.Display Omitted
