Cryopreservation and molecular genetics define advanced strategies for the conservation of endangered strains of Marble Trout Salmo marmoratus

Objective: Restocking nonnative strains of Brown Trout Salmo trutta into native Marble Trout S. marmoratus habitats has caused hybridization and a demographic decline in wild populations of the endemic species. To address this, genetic characterization of Marble Trout breeders is vital for selecting individuals for reproductive strategies. Integration of genetic analyses with sperm cryopreservation further enhances broodstock quality. Methods: In this study, a morphocellular evaluation was conducted to create a live gene bank of breeders for maintaining high-quality offspring production. A total of 229 Marble Trout males from three river basins (Adige, Brenta, and Piave rivers) were sampled at Veneto regional hatcheries in northern Italy. Genetic differentiation among populations was confirmed using a mitochondrial marker (mitochondrial DNA control region [D-loop]) and nuclear markers (lactate dehydrogenase gene LDH-C1* and microsatellites). Fertility parameters, including sperm motility and concentration, were monitored during the reproductive season (November-February) to select high-quality milt for cryopreservation. Results: Results showed that 65.49% of samples had high sperm motility throughout the season. Fewer cases of asthenozoospermia occurred mid-season (December-January) compared to the start or end of the season. Average sperm concentration was 14.95 x 10(9) cells/mL. Conclusions: Cryopreserved semen from genetically diverse Marble Trout breeders was used for artificial fertilizations. This combined approach of molecular genetics and innovative cryotechnology supports conservation efforts for Marble Trout and highlights its potential for managing endangered populations effectively. Lay summary: To protect endangered Marble Trout from hybridization, we selected breeders by using genetic analysis and preserving high-quality sperm through freezing. In a study of 229 males, we found strong fertility mid-season. This combined approach supports conservation and healthy wild populations.