Periodic Reporting for period 1 - CRYO-FISH (Gamete and embryo cryobanking for the preservation of threatened endemic fish)
Berichtszeitraum: 2021-07-01 bis 2023-06-30
Populations of fish species endemic to the Iberian Peninsula have been declining since the mid-20th century due to several factors such as habitat loss, water quality deterioration or presence of alien fish species. Furthermore, with the tendency for global warming, the typical summer droughts in Iberia are expected to become more intense and extended in time, so this fact could pose an additional extinction risk factor for the native fish. In this regard, from the 26 native species of the Portuguese freshwater fish, around 70% are already under threat according to the IUCN.
CRYO-FISH will progress on basic knowledge of the reproductive biology of endemic freshwater ichthyofauna, assessing for the first-time gamete quality throughout the breeding season. This project involves 4 species catalogued as “endangered” and “critically endangered” by the IUCN at Global & European level: Algarve nase (Iberochondrostoma almacai), Iberian minnowcarp (Anaecypris hispanica), Western riuvaco (Achondrostoma occidentale) and Torgal chub (Squalius torgalensis). New techniques for gamete assay and protocols for cryopreservation will the purpose of creating a genetic resource bank for biodiversity preservation.
This proposal is clearly in line with i) the objectives of the Horizon 2020 actions, section “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”; and ii) under the pillar of the new Horizon Europe called “Food, Bioeconomy, Natural Resources, Agriculture And Environment”.Therefore, any action aimed at generating knowledge of the reproductive biology of these species will be a fundamental piece for their future management and conservation and could complement the in situ and ex situ actions. The results will be of interest for other Mediterranean countries in which similar freshwater species are catalogued in the IUCN Red List (i.e. V. hispanica in Spain, A. albidus in Italy, etc.), which could use similar biotechnological tools developed over this project.
CRYO-FISH will progress on basic knowledge of the reproductive biology of endemic freshwater ichthyofauna, assessing for the first-time gamete quality throughout the breeding season. This project involves 4 species catalogued as “endangered” and “critically endangered” by the IUCN at Global & European level: Algarve nase (Iberochondrostoma almacai), Iberian minnowcarp (Anaecypris hispanica), Western riuvaco (Achondrostoma occidentale) and Torgal chub (Squalius torgalensis). New techniques for gamete assay and protocols for cryopreservation will the purpose of creating a genetic resource bank for biodiversity preservation.
This proposal is clearly in line with i) the objectives of the Horizon 2020 actions, section “Food security, sustainable agriculture and forestry, marine and maritime and inland water research and the bioeconomy”; and ii) under the pillar of the new Horizon Europe called “Food, Bioeconomy, Natural Resources, Agriculture And Environment”.Therefore, any action aimed at generating knowledge of the reproductive biology of these species will be a fundamental piece for their future management and conservation and could complement the in situ and ex situ actions. The results will be of interest for other Mediterranean countries in which similar freshwater species are catalogued in the IUCN Red List (i.e. V. hispanica in Spain, A. albidus in Italy, etc.), which could use similar biotechnological tools developed over this project.
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”
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”
CRYO-FISH project progressed on the basic knowledge of the reproductive biology of the endemic freshwater ichthyofauna, assessing for the first-time gamete quality throughout the breeding season of several endangered native species, and developing new techniques and protocols for making a GRB for preserving the biodiversity. From the point of view of basic research, CRYO-FISH revealed unknown aspects of breeding cycle of some of the most endangered leuciscids from the Iberian Peninsula, providing data that will help ex situ conservation captivity programs. Data systematically collected and analysed produced knowledge about the right timing to collect gametes and how to manage them to assure artificial propagation of species in captivity. This action is extremely important for further restoration activities in the wild. On the other hand, project laid the groundwork for making a genetic resource bank of the most endangered fish species at national level. New gamete cryopreservation protocols were designed for the fish target species, making possible having a wild-phenotypic backup of each species. Finally, it was not possible to approach a novel technique located on the frontier of scientific knowledge: fish embryo cryopreservation. The project finish earlier than expected and the last goal was not possible to reach.
Summing up, this proposal combined the study of unknown aspects of the reproductive biology of endangered fish with the use of cutting-edge cryo-technologies, opening the door for aiding and improving the ex-situ conservation programs using innovative strategies. The generated knowledge by CRYO-FISH could be applied in other fish species both for aquaculture, research (germ lines), and conservation (biobanking) purposes.
Summing up, this proposal combined the study of unknown aspects of the reproductive biology of endangered fish with the use of cutting-edge cryo-technologies, opening the door for aiding and improving the ex-situ conservation programs using innovative strategies. The generated knowledge by CRYO-FISH could be applied in other fish species both for aquaculture, research (germ lines), and conservation (biobanking) purposes.