The effect of ovarian fluid on the motility of cryopreserved sperm of native Mediterranean Trout (S. macrostigma)

In recent decades, native salmonid species have been the focus of major conservation projects due to the sharp decline in populations throughout their whole distribution area. In this regard, the project “LIFE” Nat.Sal.Mo funded by the EU, aims to ensure the recovery and the conservation of native Mediterranean trout (S. macrostigma = S. cettii) in Molise river basins (Molise region-Southern Italy). The Mediterranean trout is an endemic salmonid in Italian rivers and is currently listed on the Italian IUCN (International Union for Conservation of Nature) Red List as a “critically endangered” species, because of the of anthropogenic activities such as river pollution, poorly regulated fishing and hybridization caused by the introduction of non-native stocks. In Nat.Sal.Mo, the cryopreserved sperm in combination with appropriate fertilization schemes are used for artificial reproduction (AR) in order to maximize the genetic variability of offspring, thus safeguarding this species. In AR the control of the reproductive microenvironment is a key factor for the successful fertilization of eggs. This microenvironment includes gametes (sperm and oocytes), their respective biological fluids (seminal plasma and ovarian fluid) and a fertilization solution (basic saline solution i.e D-532) is generally used to boost fertilization rates as reported in the literature (Billard et al.,1992). Therefore, focusing on the fact that sperm motility is fundamental to achieve an efficient fertilization rate of eggs special attention must be given to the activation or fertilization medium used in artificial reproduction practices. In particular, ovarian fluid (OF) has also been shown to have a direct impact on the outcome of fertilization by modifying the performance of the sperm (velocity, duration and trajectory of movement) in different fish species. The aim of this investigation is to assess the potential effect of OF on sperm motility parameters of cryopreserved Mediterranean trout semen by comparing it with fertilization solution (FS D-532) and their combination (50% OF+ 50% FS). The rationale of this research is hence to identify the best activation medium to maximize reproductive success in AR trying to recreate a reproduction environment that is as close as possible to the natural one. The trial was conducted using eight native breeders. The semen samples (n= 7 native males) were frozen using the protocol reported by Rusco et al. (2020), whilst the OF was obtained from eggs of one female that was stripped by gentle abdominal massage. The frozen semen was thawed at 40°C for 5 s and some aliquots were diluted (ratio of 1:30) to be activated respectively with 1) OF 100%; 2) OF 50%+ 50% FS; 3) FS 100%. Sperm motility parameters were measured by computer-assisted sperm analysis (CASA). Within each treatment, the data obtained were subjected to analysis of variance (ONE-WAY-ANOVA) and differences among treatments were compared utilizing the Scheffe’s test. Significance was set at p < 0.05. The results showed a significant increase of total motility and duration of movement (longevity) using 100% OF and 50% OF + 50% FS in respect to FS alone. On the contrary, higher values (p<0.05) of the linear movement of sperm (straight-line velocity [VSL], straightness [STR] and linearity [LIN]) were obtained with FS. Furthermore, a significant increase of the amplitude of lateral head displacement (ALH) was recorded when the semen was activated in OF 50% + 50% FS compared to FS alone. In conclusion, our in vitro results showed that the suitable reproductive microenvironment for the Mediterranean trout in order to maximize the success of egg fertilization during artificial fertilization was the solution consisting of 50% OF and 50% of FS, confirming the beneficial effect of both of them as reported in literature (Dietrich et al.,2005, 2008). However, further studies are needed to evaluate a dilution rate lower than 50% in order to recreate the natural reproduction environment as much as possible and overall to validate the fertilizing ability in vivo.