The Mediterranean brown trout is currently facing a critical situation as one of the most endangered freshwater species. A complex interplay of climate change and human activities has inflicted significant damage to its biodiversity. The introduction of alien trout is one of the most serious threats to native populations’ intraspecific diversity. In Molise region (south-Italy) the conservation project LIFE Nat.Sal.Mo aims to restore the genetic integrity of native Mediterranean trout. In this regard, we used two main approaches to efficiently reduce genetic introgression in the native trout populations within the project area: • to only permit the access of not-introgressed wild breeders to the main natural spawning grounds; • to produce native eyed eggs through artificial reproduction with frozen semen of native wild males. Frozen semen combined with proper fertilization schemes was used in artificial reproduction to increase the genetic variability of offspring. Regarding the second point, establishing a semen cryobank with high genetic variability was a crucial step in the Nat.Sal.Mo project to safeguard the existing genomic diversity of native trout populations inhabiting the Molise rivers. Therefore, we developed an effective semen freezing protocol to establish the first European sperm cryobank supporting the conservation and restocking activities for Mediterranean brown trout. The semen cryobank preserved the natural genetic variation by creating a source of genetic material from representative populations of Mediterranean brown trout in the Biferno and Volturno rivers. Approximately 2,091 semen doses from 161 native breeders were stored in the cryobank, providing a valuable ""tank"" of genetic variability for artificial supportive breeding. The use of frozen semen avoided transferring wild fish into an artificial environment, which frequently causes significant loss by stress or domestication: semen stripping from native male to be frozen as well as the fertilization with cryopreserved semen was performed on the riverbed. The results achieved during the project were highly satisfactory: 88 native females were used to obtain 305,000 eggs, which were fertilized with 346 semen doses from 161 native males for a total of 346 unique male x female crosses to increase the genetical variability thus maintaining a high fitness within selfsustaining populations. The fertilized eggs were incubated in two hatcheries, one for each basin, with an average fertility rate of 75%. Around 230,000 eyed eggs were produced and stocked using nesting techniques in suitable sites chosen for the native trout restocking program. Nesting techniques proved to be a more effective approach by reducing non-adaptive selective pressure compared to exposing larvae and early life stages to an artificial environment. Significant progress was made in reducing genetic introgression (from 0.22 at the project's start to 0.05 at the end of the project) and increasing native trout populations (from 70% of the starting situation to 94% at the end). These results are encouraging as low levels of introgression and inbreeding are essential to prevent detrimental effects on fitness-related traits that could jeopardize population survival. The semen cryobank not only contributed to the project's objectives but also create opportunities for supporting hatchery management of Mediterranean brown trout at EU level and other endangered salmonid species, preserving the wild diversity of native populations. Breeding in captivity can lead to the loss of genetic variability and ""wildness" in subsequent generations, potentially putting populations at risk of extinction. Soon, the implementation of a native broodstock is expected to restock the watercourses of the Molise region with only autochthonous materials, and the remaining semen doses in the cryobank will play a pivotal role in ensuring the sustainability of the project's objectives even after the project’ end. In conclusion, the innovative techniques employed in the Nat.Sal.Mo project, such as semen cryopreservation and nesting, offer the opportunity to develop new plans for the restoration of other target species and contexts. However, to ensure the sustainability of conservation measures beyond the project's end, participatory governance tools such as the two stipulated River Contracts are being adopted. These tools will guarantee the continuation of habitat restoration, fluvial reconnection, genetic introgression reduction, and further increase of native trout.
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