During the spring of 2022 several endangered leuciscid species (A. hispanica, S. aradensis, A. occidentale and I. lusitanicum) were sampled both at the Vasco da Gama aquarium facilities and in some rivers of the Algarve region (Portugal). Sperm samples were extracted by mild abdominal pressure and sperm motion parameters were assessed for the first time in four species, using a computerised analysis system. The results obtained showed that spermatozoa kinetic patterns were similar for all 4 species, with high motility and velocity values after the sperm activation time and with a marked decrease after 20 seconds. On the other hand, sperm longevity was highly variable between species, with short longevities (around 40 s) for A. hispanica and S. aradensis, and longer longevities (100-120 s) for A. occidentale and I. lusitanicum. At the same time, morphometric analysis was carried out for the four target species, revealing that spermatozoa showed similar sizes and shapes to other external fertilizers belonging to Leuscididae, with small spherical heads, uniflagellate and without acrosomes.
In addition, different short-term gamete storage trails were performed by diluting sperm in 1:9 (sperm:extender) and storing them at 4ºC. Although the results obtained were uneven among the species studied, the dilution and extender used generated motilities above 40% up to day 4 of storage in S. aradensis and I. lusitanicum, and up to day 1-2 in A. hispanica and A. occidentale, respectively. Finally, gamete cryopreservation trials were also carried on these threatened species. Although cryopreserved samples showed significantly lower motility than fresh samples, some protocols generate acceptable percentages of viability, DNA integrity, and sperm motility in some species such as I. lusitanicum and A. occidentale. The data revealed that the protocol based on 10% DMSO plus 7.5% egg yolk generated the best results.
This study is the first to assess the reproductive traits of wild and captive populations of endangered leuciscids endemic from the Iberian Peninsula, describing the spermatozoa kinetics and developing protocols for managing male gametes both in short- and long- term storage. Outcomes will provide new and useful tools to complement the management and conservation of ex-situ breeding programs that are being developed on these four endangered species.
Regarding publications, the project has generated 5 communications to Workshop/Congresses; 3 manuscripts in ISI journals born directly from the project (1 published and 2 submitted); 1 Master Thesis codirected; 5 manuscripts born from CCMAR collaborations (1 published and 4 submitted); and 3 non-scientific publications]. Regarding dissemination, during the project the researchers have organizaed many activities such as i) outreach days focused on fish & rivers conservation: ii) the participation on conferences & international workshops; and finally iii) the organization of the “Workshop on Reproductive Biotechnology & Cryobanking in Aquatic Species”
