The first semen cryobank in Europe for the protection of Mediterranean brown trout wild populations: the results over four years within the LIFE Nat.Sal.Mo project (Molise region. South Italy)

The implementation of the first European semen cryobank of Mediterranean brown trout (S. cettii) was made possible by the LIFE Nat.Sal.Mo project, which aims to protect and restore the genetic integrity of the native populations of this priceless spe-cies that is on the verge of extinction. The interaction of different climate and human threats have severely harmed its biodiversity over the decades, however the introduction of alien trout repre-sented one of the most serious threats to the native populations’ intraspecific diversity. The main usefulness of the present cryo-bank is represented by its practical application to support the artificial reproduction activities aimed to maximize the genetic variability of the offspring and reduce the genetic introgression rate in the native populations of the project area.From a practical point of view, developing an effective semen freezing procedure was the first step in setting up the cryobank. The fine-tuned cryopreservation protocol allowed for the storage within the cryobank of 1683 semen doses, from 150 native breed-ers. During the four years of the project, about 30% of these semen doses were used for artificial fertilization purposes. The present activities were accomplished directly on the riverbank by dividing the eggs from each native female into equal aliquots and fertilizing each of them with frozen semen from different males, following a cross-fertilization matrix scheme in order to increase the genetic variability of the offspring. Subsequently, the fertil-ized eggs were incubated at the hatchery until they reached the eyed stage. In order to elude the domestication of wild stock, the restocking of the suitable areas was carried out using eyed eggs with the ‘artificial nesting’ method. This technique assured a successful approach, therefore avoiding the exposure of larvae and early life stages to an artificial environment. This innovative technique resulted in excellent fertilization rates in vivo, which ranged from 64% to 81%, confirming also the efficiency of the sperm freezing protocol used and increase in the percentage of native specimens in the project's areas.The remaining genetic resources of the cryobank represent sup-portive material both to ensure the sustainability of the project in the future, and to play a powerful multiplying effect which will affect other river basins at national and European levels where S� cettii populations are at risk of extinction.