The Use of Ovarian Fluid as Natural Fertilization Medium for Cryopreserved Semen in Mediterranean Brown Trout: The Effects on Sperm Swimming Performance

Simple Summary In the context of the "Life-Nat.Sal.Mo" project, obtaining an effective semen cryopreservation protocol was an important milestone that allowed the implementation of the first European cryobank of native Mediterranean brown trout (S. cettii) inhabiting Molise rivers (Italy). The main use of our semen cryobank is represented by its practical application in artificial reproduction practices aimed at maximizing the genetic variability of the offspring and reducing the genetic introgression in the native trout populations in the project area. Thus, the choice of the most suitable activation/fertilization medium represents the last key step in the development of artificial reproduction protocols using cryopreserved semen. Therefore, the aim of the present study was to investigate the effect of ovarian fluid as a natural activation media of spermatozoa on the post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid. Our results suggest that the presence of ovarian fluid alone or in combination with D-532 in the artificial microenvironment of reproduction represents a key factor to increase the success of fertilization when the frozen semen of Mediterranean brown trout is used. D-532 fertilization solution is generally used to replace the water or ovarian fluid during artificial reproductive practices in salmonids due to its ability to boost sperm motility and increase fertilization rates compared with natural activation media. However, the maintenance of ovarian fluid in a reproductive microenvironment gives it the advantage of protecting the eggs from potential harmful factors from the external environment and simplifying the field operations related to its removal when D-532 is used alone. In light of this, the aim of the present study was to investigate in vitro, for the first time, the effect of ovarian fluid (OF 100%) on post-thaw sperm swimming performance of Mediterranean trout, comparing it with D-532 and a mixed solution of 50% D-532 and 50% ovarian fluid (OF 50%). The percentage of motile spermatozoa and movement duration was significantly increased in OF 100% and OF 50% compared with D-532. Sperm velocity was higher in D-532, but significant differences were recorded only with OF 100%. In conclusion, these results suggest that the presence of ovarian fluid alone or in combination with D-532 in an artificial microenvironment of reproduction represents a key factor in potentially increasing fertilization success when the frozen semen of Mediterranean brown trout is used.