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- Assessment of male reproductive traits in endangered leuciscids from the Iberian Peninsula: first attempts to store gametes both at short- and long-termPublication . Hernandez, Ana; Sousa-Santos, Carla; Gil, Fátima; Cabrita, Elsa; Guerreiro, Pedro; Gallego, VictorDuring the spring of 2022, several endangered leuciscid species (Anaecypris hispanica, Squalius aradensis, Anachondrostoma Occidentale, and Iberochondrostoma 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 gentle abdominal pressure and sperm motion parameters were assessed for the first time in four species, using a computerized 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. 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, which could indicate a latitudinal pattern in terms of sperm longevity. 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, a short-term gamete storage trail was 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 days 1-2 in A. hispanica and A. occidentale, respectively. Finally, gamete cryopreservation trials were also carried out 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 for these four endangered species.
- Sperm characterization of endangered leuciscids endemic from the iberian peninsula: gamete storage as a tool for helping ex-situ breeding programsPublication . Hernandez, Ana; Sousa-Santos, Carla; Gil, Fátima; Guerreiro, Pedro Miguel; Cabrita, Elsa; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and several types of actions (from in situ to ex situ measurements) have been applied over the past decades for preserving these species. However, limited knowledge about their reproductive biology makes it necessary to investigate different aspects of the reproductive cycle for improving breeding programs. The main objectives of this work were to advance knowledge concerning sperm kinetics and spermatozoa morphology and to develop protocols for the short- and long- term storage of gametes. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum, Achondrostoma occidentale, and Squalius aradensis) were sampled during the spring of 2022 both in captive populations kept at Aquário Vasco da Gama (AVG) and in wild populations from different Portuguese rivers. Sperm samples were collected and sperm motion parameters were assessed, for the first time, for these four species. Sperm kinetics differed between species in motility and velocity traits, also showing a different number of sperm subpopulations. The longevity of sperm (swimming period) was also different among species: the shortest period was obtained for the wild population of S. aradensis (values close to zero at 40 s), and the longest swimming period for the captive population of I. lusitanicum (values close to zero at 120 s). Furthermore, different storage trials were carried out diluting the sperm in a extender solution (75 mM NaC1, 70 mM KC1, 2 mM CaC12, 1 mM, MgSO4, l0 mM Hepes, pH 8) at a ratio 1:20 (sperm:extender). Sperm quality (>40% of motile cells) was kept for a maximum of four days of storage, depending on the species. In addition, new cryopreservation protocols (using DMSO, Methanol and/or egg yolk) were tested for cryobanking the sperm of these threatened species. Cryopreserved samples showed significantly lower motility when compared with fresh samples, and the best results were obtained for I. lusitanicum, reaching 20% of motile cells after thawing using 10% of DMSO supplemented with 10% of egg yolk. This study is the first of its kind to successfully achieve gamete cryopreservation of Iberian endemic and endangered freshwater fish species, developing new and useful tools to complement the management and conservation programs.
- Assessment of the reproductive status of captive populations of endangered leuciscid species from the iberian peninsula: a. hispanica, I. lusitanicum and a. occidentalePublication . Hernandez, Ana; Gil, Fátima; Sousa-Santos, Carla; Cabrita, Elsa; Guerreiro, Pedro Miguel; Gallego, VictorPopulations of freshwater fish species endemic to the Iberian Peninsula have been declining since the mid-20th century, and the captive breeding of highly endangered species is considered to be a useful tool to restock declining populations. A pioneer project of supportive breeding of critically endangered fish started in 2007 at the Aquário Vasco da Gama (AVG), and this work aims to show the reproductive status of the breeders which make up the current captive broodstoks. Populations of different endangered leuciscid species (Anaecypris hispanica, Iberochondrostoma lusitanicum and Achondrostoma occidentale) were sampled at AVG during the spring of 2022. Breeders were counted and sexed, and males were stripped to check for the presence of spermatozoa. The sperm volume was assessed visually, and spermatozoa motility was assessed by a CASA system. Sperm samples were classified into four classes based on the percentage of motile cells: C-I ≤ 25%, C-II = 25–50%, C-III = 50–75%; and C-IV > 75%. The captive population of A. hispanica consisted of 63 individuals and showed a 40% of spermiating males, with an average volume of 5–10 µL. The histogram of sperm quality reported that 15% males had sperm motility of C-II, 50% of males had sperm motility of C-III and, finally, 35% of males had sperm with the high-quality class (C-IV). The population of I. lusitanicum consisted of 599 individuals and showed 93% of spermiating males, with an average volume of 15–20 µL. The histogram of sperm quality reported that most part of the males had good sperm quality belonging to C-III and C-IV class (26% and 71%, respectively), while just 1 male showed bad quality sperm (C-II). The captive population of A. occidentale consisted of 193 individuals, showing a 62% of spermiating males with an average volume of 20–25 µL. The histogram showed that 6% males had sperm motility of C-I, 26% of males had sperm motility of C-II, the most part of the males (45%) showed a sperm quality of C-III and, finally, 23% of males had sperm with the high-quality class (C-IV). Since the project began in 2007, more than 12,000 fish of these three critically endangered species have been released to restock the populations from which the respective wild breeders were caught. All captive fish were released after a maximum of three consecutive generations in captivity, and new stocks were established with wild adults from the target populations, to avoid the negative effects of inbreeding and genetic drift on the original genetic pool.