Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males. After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure. Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations
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