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[EN] Fish sperm motility is nowadays considered the best biomarker for the quality of fish spermatozoa, and sperm motion parameters from more than 300 fish species have been reported in more than 1500 scientific articles covering a wide range of topics, from molecular biology to ecology. The most studied topics have been (i) the sperm storage (involving both the use of chilled¿storage protocols for short¿term periods and sperm cryopreservation techniques for long¿term storage), (ii) the sperm physiology (fathom in the spermatozoa activation process and the whole propulsion machinery of the sperm cells) and (iii) the broodstock management (covering aspects such as rearing conditions, dietary requirements or hormonal induction treatments). In addition, other aquaculture and ecological topics, such as (iv) the knowledge of the breeding cycle of the species, (v) the phenomenon of the sperm competition and (vi) ecotoxicological studies for the evaluation of aquatic environments, have also been approached from the evaluation of sperm motion performance. Therefore, fish sperm motility assessment can serve as a potential tool for aquaculture and ecological purposes, covering key topics of fundamental and applied research. This review gives an overview of the major research areas in which fish sperm motility has been applied successfully. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 642893 (IMPRESS). VG has a postdoc grant from the UPV (PAID-10-16). ; Gallego Albiach, V.; Asturiano Nemesio, JF. (2018). Fish sperm motility assessment as a tool for aquaculture research, a historical approach. Reviews in Aquaculture (Online). 1-28. https://doi.org/10.1111/raq.12253 ; S ; 1 ; 28

[EN] Although a relatively high number of sperm quality biomarkers have been reported over the years in several fish species, sperm motility is nowadays considered the best biomarker for fish spermatozoa. The first scientific reports focusing on fish sperm motility date from a century ago, but the objective assessment allowed by computer-aided sperm analysis (CASA-Mot) systems was not applied to fish species until the mid-1980s. Since then, a high number of sperm kinetic parameters from more than 170 fish species have been reported in more than 700 scientific articles, covering a wide range of topics, such as sperm physiology, sperm storage, broodstock management, the phenomenon of sperm competition, ecotoxicology and understanding the life cycle of the species. The sperm kinetic parameters provided by CASA-Mot systems can serve as powerful and useful tools for aquaculture and ecological purposes, and this review provides an overview of the major research areas in which fish sperm motility assessment by a CASA-Mot system has been used successfully. ; This writing of this manuscript as received funding from the European Union's Horizon 2020 Research and Innovation program under the Marie Sklodowska-Curie Grant Agreement No. 642893 (ETN IMPRESS). V. Gallego has a postdoctoral grant from the Universitat Politecnica de Valencia (PAID-10-16). ; Gallego Albiach, V.; Asturiano Nemesio, JF. (2018). Sperm motility in fish: technical applications and perspectives through CASA systems. Reproduction Fertility and Development. 30(6):820-832. https://doi.org/10.1071/RD17460 ; S ; 820 ; 832 ; 30 ; 6

[EN] Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool for ex situ conservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a nonpermeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful for ex situ conservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity. ; Study partially funded by the Fundacion Biodiversidad (PRCV00683). PG-S has a contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ACIF 2018 (ACIF/2018/147). VG has a postdoc contract from ...

[EN] Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH(i)) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pH(i) range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH(e)), as well as between the pH(i) and the pH of the diluent. The change post-activation in seawater (ASW) depended on the initial pH(e) of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH(e) 8.0; an acidification when pH(e) > 8, and no pH(i) variation when pH(e) was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH(i) changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. (C) 2020 Elsevier Inc. All rights reserved. ; Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS). Victor Gallego has a postdoc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-2017-34200 ; Pérez Igualada, LM.; Gallego Albiach, V.; Asturiano, JF. (2020). Intracellular pH regulation and sperm motility in the European eel. Theriogenology. 145:48-58. ...

[EN] Fish sperm motility is nowadays considered the best sperm quality biomarker in fish, and can be evaluated both by subjective and computerized methods. With the aim to compare the precision and accuracy of both techniques, fish sperm samples were assessed by subjective methods and by a computerassisted sperm analysis (CASA-Mot) system, and simultaneously by three different technicians with different degrees of expertise on the sperm quality analysis. Statistical dispersion parameters (CV, coefficient of variation; and RG, range) were estimated in order to determine the precision and accuracy of the techniques and the influence of laboratory staff on sperm motion assessments. Concerning precision, there were not much significant differences between the technical support staff (high, medium, and low experimented technician), and statistical dispersion parameters were quite similar between them independent of the technique used and the sperm motility class analyzed. However, concerning accuracy, experimented technician reported subjective motility values very closed to the values provided by the CASA-Mot system, only 10 percentage points away from the data provided by a CASA-Mot system. However, medium and low experimented technicians often overestimate the CASA-Mot values, and amplitudes up to 30 percentage points were detected in several sperm assessments. To sum up, both the technique (subjective or objective) and the technician (degree of expertise) became key factors in order to reach accurate motility estimations, so the use of both qualified staff and novel CASA-Mot systems seems to be a critical requirement for obtaining satisfying results in fish species with similar motility patterns. ; This study is funded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 642893 (IMPRESS) and the COST Office (COST Action FA1205: AQUAGAMETE). VG has a postdoc grant from the UPV (PAID-10-16). ; Gallego Albiach, V.; Herranz-Jusdado, JG.; Rozenfeld, ...

[EN] New specific European eel (Anguilla anguilla) recombinant gonadotropins (aarGths) produced in the ovarian cells of Chinese hamsters (CHO) were used to induce maturation in captive male eels. In the first experiment, five different hormonal treatments were assayed: one group was given a constant dose of recombinant European eel follicle-stimulating hormone (aarFsh; 4 ¿g/fish) for 9 weeks, and the second group received a constant dose of recombinant European eel luteinizing hormone (aarLh; 2 ¿g/fish) also for 9 weeks. The other three groups were injected with different combinations of both aarGths (some doses constant, some variable). All five treatments stimulated androgen synthesis, but the increase was more pronounced in the fish treated with a combination of both aarGths. Unlike aarLh, aarFsh alone was able to induce spermiation, the best results were achieved in the fish that were treated with a constant dose of aarFSH and an increasing dose of aarLH, with spermiation being induced (20% motile cells) despite the fact that these fish were immature at the start of the experiment. In order to improve sperm quality, a second experiment was performed. Immature males received three constant doses of aarFsh (2.8, 1.4 or 0.7 ¿g/fish) and increasing doses of aarLh (every 3 weeks; 1, 2, 6 ¿g/fish). All the treatments induced spermiation, however the best sperm quality (with ¿50% motile cells) was observed in the males treated with the highest dose of aarFsh. In conclusion, these specific recombinant gonadotropins have demonstrated their capacity to induce spermatogenesis and spermiation in vivo in a teleost fish, the European eel. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (EN IMPRESS), the Spanish Ministry of Economy, Industry and Competitiveness (Project REPRO-TEMP; AGL2013-41646-R), Generalitat Valenciana (PROMETEOII/2014/051), VLC/CAMPUS Program (SP20140630) and the COST Office (COST ...

[EN] The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla). In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5¿mL, which is useful for larger scale fertilization trials. The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs. ; Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement N 642893 (IMPRESS), including the JGHJ and CR pre-doctoral contracts. MM has a postdoc grant from the UPV (PAID -10-18). VG has a postdoc grant from the MICIU (Juan de la CiervaIncorporacion; IJCI-2017-34200). This research was supported by the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities of Hungary within the framework of water related researches of Szent Istvan ...

210 215 133 ; S ; [EN] The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla). In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5¿mL, which is useful for larger scale fertilization trials. The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs. Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement N 642893 (IMPRESS), including the JGHJ and CR pre-doctoral contracts. MM has a postdoc grant from the UPV (PAID -10-18). VG has a postdoc grant from the MICIU (Juan de la CiervaIncorporacion; IJCI-2017-34200). This research was supported by the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities of Hungary within the framework of water related researches of ...

[EN] During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka¿s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation. ; This publication was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), the COST Office (COST Action FA 1205, AQUAGAMETE), and the Research Centre of Excellence -1476-4/2016/FEKUT ; Herranz-Jusdado, JG.; Kása, É.; Kollár, T.; Gallego Albiach, V.; Peñaranda, D.; Rozenfeld, C.; Pérez Igualada, LM. (2018). Handling and treatment of male European eels (Anguilla anguilla) for hormonal maturation and sperm cryopreservation. Journal of Visualized Experiments. 131(e56835):1-6. https://doi.org/10.3791/56835 ; S ; 1 ; 6 ; 131 ; e56835

[EN] It is essential to define an optimized standard method to assess the fish sperm quality to minimize the differences between the results obtained by different laboratories. Only this optimization and standardization can make them useful from academia to industry. This study presents the validation of sperm motility assessment using a CASA-Mot system for three endangered diadromous fish species: European eel (Anguilla anguilla), Atlantic salmon (Salmo salar) and Siberian sturgeon (Acipenser baerii). To attain this goal, different technical and data processing methods were tested: 1) magnification lens (×10 and ×20), 2) Spermtrack® reusable chambers (10 and 20¿¿m depth) and 3) different frame rates (50¿¿¿FR¿¿¿250). The results suggested that the sperm motility assessment for eel, salmon and sturgeon should be performed at 200, 250 and 225¿frames¿s¿1, respectively. Moreover, to obtain a high number of analysed spermatozoa in less time and a natural movement of the sperm cells, it is recommended to use ×10 objective and 20¿¿m depth. In conclusion, different technical settings influence sperm kinetic parameters and should be validated for each fish species to allow the comparison of results between laboratories. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No [642893]. AV is granted by the CONICIT and MICITT, Costa Rica. The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic projects "CENAKVA" (No. CZ.1.05/2.1.00/01.0024), "CENAKVA II" (No. LO1205 under the NPU I program), project Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370), by the Czech Science Foundation (project No. 17-19714Y). ; Caldeira, C.; Hernández-Ibáñez, S.; Valverde, A.; Martin, P.; Herranz-Jusdado, JG.; Gallego Albiach, V.; Asturiano Nemesio, JF. (2019). Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and ...

[EN] Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed. ; This study received funding from the project REPRO-TEMP (AGL2013-41646-R) funded by the Spanish Ministry of Economy and Competitiveness, and from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), which ...