Thawing rate effects on the cryosurvival of Mediterranean brown trout spermatozoa

The Mediterranean trout, Salmo cettii, is listed in Annexe II of the EU Habitats Directive and is included as ‘critically endangered’ in the Italian freshwater fish Red List. One of the most effective tools to avoid the extinction is the semen cryobank and, in this regard, the development of a successful sperm cryopreservation protocol is needed. Therefore, the purpose of the present study was to evaluate the effects of two different thawing rates on the in vitro and in vivo sperm quality of S. cettii populations of Molise. Native trout were captured by electro-fishing. Semen was collected from 40 males to obtain five total pools, each diluted 1:3 (v:v) with a freezing extender composed of 0.3M glucose, 10% DMSO and 10% egg yolk. The semen was loaded in 0.25 mL straws and equilibrated at 5 °C for 10 min. The straws were frozen through the exposure at 5 cm above the liquid nitrogen level for 10 min, dipped and stored in the liquid nitrogen. Straws were thawed at two different thawing rates 30 °C for 10 s and 10 °C for 30 s, using a water bath. The sperm parameters evaluated were: motility and movement duration, viability (SYBR-14, PI) and DNA integrity (Acridine Orange). Fertilisation trials were performed using three groups of eggs (N≃100) inseminated with: (a) fresh sperm (control group); (b) sperm thawed at 30 °C for 10 s; (c) sperm thawed at 10 °C for 30 s. The data obtained in vitro showed that the freezing process impaired the post-thaw sperm quality compared to the fresh semen (p<.05). In addition, we also found significantly higher motility and viability (p<.05) in sperm thawed at 10 °C for 30 s than the 30 °C for 10 s. The fertilisation and hatching rates were significantly higher in fresh sperm (73.26 ± 5.17 and 68.89 ± 5.51) respect to the frozen semen. However, the best eyed and hatched rates were found using semen thawed at 10 °C for 30 s (58.6 ± 2.8% and 54.5 ± 2.8%) than sperm thawed at 30 °C for 10 s (32.9 ± 4.9% and 29.9 ± 4.4%) (p<.05). In conclusion, the use of low thawing rate (10 °C for 30 s) improved the semen fertilisation ability of S. cettii. Our findings provide an important contribution for the creation of a sperm cryobank aiming at the restoration of S. cettii in Molise. Natural reproduction of native trout occurs on the spawning grounds at the main springs of Volturno and Biferno rivers (T≃10 °C). Thus, the encouraging results at the thawing temperature of 10 °C, would facilitate the on-field artificial reproduction of wild breeders, using directly the spring water.