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[ES] La anguila europea (Anguilla anguilla) es una especie con un elevado interés acuícola a nivel mundial y con una alta producción en el contexto de la acuicultura europea. Sin embargo, la piscicultura de anguilas se presenta como una actividad no sostenible en el tiempo, debido fundamentalmente a que las empresas dependen del abastecimiento de juveniles del medio natural. Además, actualmente se encuentra catalogada como especie en peligro crítico de extinción (UICN) debido a la reducción drástica de su población en las últimas décadas. Por lo tanto, la reproducción en cautividad de esta especie se presenta como una pieza clave para su conservación, lo que ayudará a reducir la presión sobre las poblaciones naturales, facilitará el abasteciendo a las anguilifactorías, y permitirá repoblaciones en zonas en las que históricamente se localizaba la anguila. El objetivo del presente estudio fue evaluar la aplicación de métodos alternativos de inducción de la maduración sexual (sistemas de liberación controlada, SLC) en hembras de anguila europea. Con la finalidad de desarrollar protocolos capaces de inducir la ovulación en anguila europea la evaluación se realizó tanto en términos biológicos como ecónomicos. Los resultados mostraron que la implantación de bombas osmóticas indujo la maduración en el del 80% de las hembras, reduciendo la duración del tratamiento hormonal en un 50% de ellas, que alcanzaron la madurez en las primeras 4-10 semanas de tratamiento. Con respecto a la evolución del peso de las hembras durante la maduración se observó un incremento en la semana previa del 16±6% en el grupo OP-2ML4 y del 20±14% en el grupo control. En cuanto al índice gonadosomático (IGS), las hembras implantadas con bombas osmóticas presentaron valores similares al grupo control, 49.5±2.8% y 51.2±9.8%, respectivamente. Asimismo, se obtuvo un total de 6 puestas alcanzando el estado de embrión formado con la cabeza, ojos y somitas distinguible como máximo desarrollo embrionario. Fi, [EN] The European eel (Anguilla anguilla) is a species with high aquaculture interest worldwide and with a high production in the context of European aquaculture. However, eel fish farming is presented as an unsustainable activity over time, mainly due to the fact that companies depend on the supply of juveniles from the natural environment. In addition, it is currently listed as a critically endangered species (IUCN) due to the drastic reduction in its population in recent decades. Therefore, the captive reproduction of this species is presented as a key element for its conservation, which will help reduce the pressure on natural populations, facilitate the supply of eel farms, and allow repopulation in areas where historically the eel was found. The objective of the present study was to evaluate the application of alternative methods of induction of sexual maturation (controlled release systems, CRS) in female European eels. In order to develop protocols capable of inducing ovulation in European eel, the evaluation was carried out both in biological and economic terms. The results show that the implantation of osmotic pumps induced maturation in 80% of the females, reducing the duration of hormonal treatment in 50% of them, which reached maturation in the first 4-10 weeks of treatment. Regarding the evolution of the weight of the females during maturation, an increase in the previous week of 16±6% in the OP-2ML4 group and 20±14% in the control group. Regarding the gonadosomatic index (GSI), the females implanted with osmotic pumps presented similar values to the control group, 49.5±2.8% and 51.2 ± 9.8%, respectively. Likewise, a total of 6 clutches were obtained, reaching the embryo state formed with distinguishable head, eyes and somites as maximum embryonic development. Finally, this study shows that the use of osmotic pumps loaded with carp pituitary extract (CPE) is an effective method to induce sexual maturation in female European eels, generating early matur

[ES] La anguila europea (Anguilla anguilla) es una especie importante para la acuicultura europea, siendo muy apreciada tanto en el mercado europeo como en el asiático. Sin embargo, su producción actual todavía se base en el engorde de anguilas capturadas en el medio natural, debido a que aún no es posible reproducir anguilas en cautividad. Además, se ha observado una disminución drástica en el número de anguilas europeas que migran desde Europa y el norte de África a los lugares de puesta en el Océano Atlántico, lo que ha llevado a que la especie se incluya en la lista roja de la UICN como en peligro crítico de extinción. Por tanto, la cría en cautividad se postula como la única sostenible para preservar esta especie i) reduciendo la presión sobre las poblaciones naturales, ii) facilitando el suministro a las granjas acuícola, y iii) permitiendo la repoblación en zonas donde históricamente se encontraba distribuida esta especie. El objetivo de este estudio fue evaluar la aplicación de métodos alternativos (bombas osmóticas ALZET®) para la inducción de la maduración en machos de anguila europea. Estos sistemas se evaluaron tanto en téminos biológicos como económicos, con la intención de diseñar protocolos capaces de inducir la espermatogénesis en esta especie. Los resultados mostraron que la implantación de bombas osmóticas ALZET 1004 (grupo OP-100) y ALZET 2006 (grupo OP-200), indujo la maduración y producción de esperma en machos de anguila europea. En cuanto al índice gonadosomático (IGS), los grupos implantados con bombas osmóticas alcanzaron valores de 2.23 ¿ 0.81 (OP-100) y 3.06 ¿ 1.43 (OP-200), sin embargo, en general, todos estos valores se encontraron por debajo de los resultados obtenidos mediante tratamientos a base de inyecciones semanales de hCGrec (grupo Control). En cuanto a la producción del esperma, los grupos experimentales (OP-100 y OP-200) alcanzaron volúmenes de hasta un 1 mL/100 g pez, densidades de hasta 5 x 109 (OP-200) y 10 x 109 espermatozo, [EN] The European eel (Anguilla anguilla) is an important species for European aquaculture, being highly appreciated both in the European and Asian markets. However, its current production still consists in the fattening of eels captured in the natural environment, due to the fact that it is not yet possible to reproduce eels in captivity. In addition, a drastic decrease has been observed in the number of wild European eels migrating from Europe and North Africa to the spawning sites in the Atlantic Ocean, leading to the species being included in the IUCN red list as critically endangered. Therefore, breeding in captivity is postulated as a key alternative in order to save this species, which will help to reduce the pressure on natural populations, it will facilitate the supply to the eel farms, and it will allow repopulation in areas where those that historically were located the eel. The objective of this study was to evaluate the application of alternative methods (ALZET® osmotic pumps) for the induction of maturation in male European eel. These systems were evaluated both in biological and economic terms, with the intention of designing protocols capable of inducing spermatogenesis in this species. The results showed that the implantation of osmotic pumps ALZET 1004 (group OP-100) and ALZET 2006 (group OP-200) induced the maturation and production of sperm in male European eels. Regarding the gonadosomatic index (IGS), the implanted groups with osmotic pumps reached values of 2.23 ¿ 0.81 (OP-100) and 3.06 ¿ 1.43 (OP-200), however, all these values were below the results obtained by treatments based on weekly injections of hCGrec (Control group). Regarding sperm production, the experimental groups (OP-100 and OP-200) reached volumes of up to 1 mL / 100 g fish, densities of up to 5 x 109 (OP-200) and 10 x 109 sperm / mL , and low percentages of motility of approximately 10% of mobile cells. Finally, from an economic point of view, the cost of osmotic pumps was ver

[ES] En las tres últimas décadas la anguila europea (Anguilla anguilla) ha experimentado una disminución de hasta un 99% en su captura en varias de las zonas que forman parte de su área de distribución geográfica, por lo que la cría en cautividad es considerada hoy en día una práctica clave para salvar a esta especie. En este sentido, obtener gametos de alta calidad es fundamental, así como avanzar en el estudio de nuevos tratamientos hormonales que tienen como finalidad mejorar los métodos actuales. Debido a esto, el objetivo de este estudio de investigación es, primero, evaluar el efecto de dos tratamientos hormonales (OVI, una ¿-coriogonadotropina recombinante; y VET, una gonadotropina coriónica humana purificada de orín femenino) sobre el rendimiento reproductivo de los machos de anguila europea, y tras elegir la mejor hormona, comparar los efectos de tres dosis diferentes con el fin de reducir los costes de la maduración artificial. Los resultados de este trabajo mostraron que el tipo de hormona utilizada (gonadotropina recombinante frente a purificada) afectó considerablemente a la evolución de la espermiación en los machos de anguila europea, y que la hormona recombinante (OVI) generó unos mejores resultados en términos de cantidad y calidad de esperma en la mayoría de las semanas de tratamiento, consiguiendo así un tratamiento efectivo para inducir la espermiación en esta especie. Por otro lado, en el experimento de dosis, los resultados indicaron que desde la dosis más baja hasta la más elevada (0,25 a 1,5 UI/g de pez) todos los tratamientos son capaces de inducir el proceso de espermiación completo. No obstante, fue necesaria una dosis semanal de 1,5 UI/g de pez de la hormona recombinante (OVI) para poder proporcionar una cantidad significativa (volumen y densidad) de muestras con una alta calidad (motilidad y velocidad) durante todo el tratamiento. Finalmente, el análisis económico demostró que la hormona recombinante (OVI, 1,5 UI/g de pez) tuvo una mayor, [EN] In the past three decades the European eel (Anguilla Anguilla) experienced up to 99% decline in recruitment in some parts of its distribution range, thus breeding in captivity is nowadays considered key to save this species. With this in mind, obtaining high quality gametes is fundamental, as is the ongoing study of new hormonal treatments in order to improve current methods. Therefore, the aim of this research study is to assess the effect of two hormonal treatments (OVI, a recombinant ¿-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine) on the reproductive performance of European eel males, and, after choosing the best hormone, to compare the effects of three different doses in order to cut the costs of artificial maturation. Results indicate that the type of hormone used (recombinant vs purified gonadotropins) significantly affects the progression of spermiation in European eel males, and that the recombinant hormone (OVI) produces better results in terms of sperm quantity and quality in most of the weeks of the treatment, remaining thus an effective treatment to induce spermiation in this species. On the other hand, in terms of the doses experiment, results show that from the lowest to the highest dose (0.25 to 1.5 IU/g fish) all the treatments are able to induce the whole spermiation process. However, a weekly dose of 1.5 IU/g fish of recombinant hormone (OVI) is necessary in order to provide a notable amount (volume and density) of high quality (motility and velocity) samples throughout the treatment. Finally, the economic analysis demonstrates that the recombinant hormone (OVI, 1.5 IU/g fish) has a greater profitability than the other treatments, making it possible to obtain highquality sperm for a lower price. In this context and considering the fact that in the first few weeks of any hormonal treatment there is no high-quality sperm production, long-term hormonal therapies are necessary in order to lessen the cost o

[ES] La anguila europea (Anguilla anguilla) es una especie de elevado interés pesquero y una de las especies importantes para la acuicultura mundial, pero su producción actual todavía depende de las capturas de angulas del medio natural. Catalogada actualmente como especie en peligro de extinción, la reproducción ex situ se presenta como una pieza clave para su conservación. La maduración en cautividad de esta especie requiere tratamientos hormonales largos y costosos que, en muchas ocasiones, conducen a una maduración asincrónica entre sexos. En este sentido, la optimización de los métodos de almacenamiento de esperma a corto y largo plazo pueden ser un factor clave a la hora de optimizar los procesos de fertilización in vitro. Durante el presente trabajo se llevaron a cabo una serie de experimento con el objetivo de i) mejorar los protocolos existentes de conservación de gametos a corto plazo; y de ii) optimizar los protocolos de criopreservación utilizados en la actualidad. Para el experimento de almacenamiento a corto plazo se probaron diferentes ratios de dilución (1:9 versus 1:49), diferentes temperaturas de almacenamiento (4 °C versus 20 °C), y mantener la muestra en agitación o sin agitación. Las muestras se almacenaron durante 7 días y se evaluaron distintos parámetros cinéticos a diferentes tiempos. Los resultados mostraron que la calidad del esperma no mostró diferencias significativas durante las primeras 24 h entre ambos ratio de dilución. Sin embargo, el ratio de dilución 1:49 mostró mejores resultados en tiempos de almacenamiento superiores. En este sentido, los resultados sugieren que para una conservación mayor de 48 horas es recomendable diluir el esperma a 1:49 y conservarlo a una temperatura de 4 °C. En los experimentos de criopreservación, se evaluaron diferentes viales de congelación (pajuelas versus criotubos), diferentes rampas de congelación y descongelación y, finalmente, diferentes combinaciones de crioprotecotres (methanol, FBS, BSA y yem, [EN] European eel (Anguilla anguilla) is a species of high fishing interest and one of the most important for worldwide aquaculture. However, its current production still depends on the capture of elvers from the wild. Currently catalogued as an endangered species, ex situ reproduction is presented as a key element for its conservation. Maturation in captivity of this species requires long and costly hormonal treatments that, in many cases, lead to an asynchronous maturation between sexes. In consequence, optimization of sperm short-term and long-term storage methods can be a key factor for improving in vitro fertilization processes. During the work, some experiments were carried out with the objective of i) improving the existing short-term gamete conservation protocols; and ii) optimizing the cryopreservation protocols currently used. For the short-term storage experiment, samples were diluted at different ratios (1:9 versus 1:49), were at different temperatures (4 °C versus 20 °C) and were kept in constant agitation or still. Samples were stored for 7 days and different kinetic parameters were evaluated at different times. The results showed that the quality of the sperm did not show significant differences during the first 24 h between both dilution ratio. However, the 1:49 dilution ratio showed better results in longer storage times. In this sense, the results suggest that for longer conservation than 48 hours it is desirable to dilute the sperm at 1:49 and keep it at a temperature of 4 ° C. In the cryopreservation experiments, different freezing vials (straws versus cryotubes), different freezing and thawing ramps and, finally, different combinations of cryoprotectants (methanol, FBS, BSA and egg yolk) we evaluated. The combination of methanol (10%) and egg yolk (5%) as a freezing medium generated the best results after thawing, and some samples reached values of more than 50% of motility. Also, freezing in large volume vials (2 and 5 ml cryotubes) was also va

[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.

[EN] The sensitive state of conservation of several endemic fish species such as Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica) has led governments to consider the implementation of conservation measures to preserve their populations. However, limited knowledge about the reproductive biology of these species makes it necessary to investigate different aspects of their reproductive cycle. In this sense, the main objectives of this work were i) to advance knowledge of the breeding biology of both species, and ii) to develop protocols for the conservation of gametes for the future management and conservation. During the spring of 2019 a temporal series of samplings were carried out in different places in the Comunitat Valenciana. Sperm samples were collected and sperm motion parameters were assessed for the first time in both species. Kinetic patterns were similar showing high motility and velocity values during the first 30 s, and a rapid decrease from that point. At the same time, an in-depth morphometric analysis was carried out using computer-assisted sperm analysis software. Spermatozoa from A. iberus and V. hispanica showed similar sizes and shapes to other external fertilizers belonging to Cyprinodontiformes, with small spherical heads, uniflagellated and without acrosomes. In addition, a new cryopreservation protocol was designed for cryobanking the sperm of these threatened species. Cryopreserved samples showed lower motility than fresh samples but reaching acceptable percentages of motile cells after thawing of around 20 and 25% (A. iberus and V. hispanica, respectively). This study is the first of its kind to successfully achieve gamete cryopreservation of these two endemic and endangered species from the Iberian Peninsula, providing new and useful tools to complement the management and conservation programs that are being developed for both species.

[EN] Sperm extraction and artificial insemination may serve ex situ conservation initiatives for threatened sharks and related species. A comparison of the reproductive anatomy of eight chondrichthyans is presented in this study, emphasizing the important differences when performing these reproductive techniques. Additionally, we show how to obtain sperm samples from both living and dead specimens using cannulation, abdominal massage, or oviducal gland extraction. These tools can improve the success of breeding programs developed in aquaria and research facilities. The chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates on the planet. Given this situation, an additional strategy for the protection of these species could be the ex situ conservation projects developed in public aquaria and research centers. Nevertheless, to increase sustainability and to develop properly in situ reintroduction strategies, captive breeding techniques, such as sperm extraction and artificial insemination, should be developed. These techniques are commonly used in other threatened species and could be also used in chondrichthyans. However, the different reproductive morphologies found in this group can complicate both processes. Therefore, a comparison of the reproductive anatomy of eight distinct chondrichthyans, with an emphasis on those important differences when performing sperm extraction or artificial insemination, is carried out herein. Sharks and chimaeras belonging to the Scyliorhinidae, Carcharhinidae, Centrophoridae, Etmopteridae, Hexanchidae, and Chimaeridae families were obtained from commercial fisheries, public aquaria, and stranding events. In addition, the process of obtaining viable sperm samples through cannulation, abdominal massage, and oviducal gland extraction is described in detail for both living and dead animals.

[EN] Many species of rays and skates are endangered, and ex situ conservation programs developed by research centers and public aquaria could improve this situation. To reproduce these species in captivity, scientists need to know how to extract their sperm and how to conduct their artificial insemination; however, the anatomical diversity of the reproductive organs of this group of animals is a handicap. A comparison of the reproductive anatomies of 11 distinct species is presented here, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. In addition, the process of obtaining sperm samples from both live and dead males is described in detail, using both cannulation and abdominal massage. The superorder Batoidea (rays, skates, and relatives), constitutes one of the most threatened group of vertebrates. Strengthening ex situ conservation programs developed in research centers and public aquaria could be a way of addressing this situation. However, captive breeding programs must be improved to prevent the capture of wild animals and to develop proper in situ reintroduction strategies. Sperm extraction and artificial insemination are two techniques commonly used in other threatened species, which could also be used in rays and the like. However, the different reproductive morphologies present within this group of animals may hamper both processes. Here, we present a comparison of the reproductive anatomies of 11 distinct batoid species, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. Both male and female animals, belonging to the Rajidae, Dasyatidae, Torpedinidae and Myliobatidae families, from the Mediterranean Sea were studied. In addition, we describe the procedure to extract sperm using both cannulation and abdominal massage, either from live or dead batoids Finally, the obtention of motile sperm recovered from the oviducal gland

[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.

[ES] La perdiz roja (Alectoris rufa) ha sufrido en las últimas décadas una notable regresión de sus poblaciones como consecuencia de varios factores, entre los que cabe destacar los cambios generados en las prácticas agrícolas. En este sentido, el abandono del cultivo de algunos cereales, como el trigo (Triticum spp.), ha generado en determinadas zonas un descenso todavía más acusado de las poblaciones de esta especie, debido fundamentalmente a que su alimentación se basa en los granos de trigo que caen al suelo tras la recolección con la cosechadora. En el coto municipal de Mosqueruela (Teruel) existen dos entidades que realizan el aprovechamiento cinegético. Por un lado, está el ayuntamiento que, siendo el titular del coto, se beneficia de algunas especies cinegéticas como la cabra montés (Capra pyrenaica) y de corzo (Capreolus capreolus), que generan un elevado beneficio económico. Por otro lado, se encuentra la Sociedad de Cazadores Virgen de la Estrella , que se encarga del aprovechamiento del resto de especies cinegéticas como la codorniz (Coturnix coturnix), la paloma torcaza (Columba palumbus) o la perdiz roja, pagando un alquiler anual al ayuntamiento por dicho servicio. Por otra parte, en el municipio existen varios agricultores que realizan tareas agrícolas para otros titulares de explotaciones o para sus propias parcelas, pero que perciben unos márgenes de beneficio muy reducidos por sus cosechas. En este sentido, este colectivo estaría dispuesto a cultivar más tierras (con determinados productos como por ejemplo, el trigo), si se les dan facilidades o ayudas económicas de alguna naturaleza. En este contexto, el objetivo del presente trabajo consiste en crear un sistema de pago por servicios ambientales (PSA) con el que conseguir que los demandantes del servicio ambiental (el ayuntamiento y la sociedad de cazadores) y los oferentes (los agricultores de servicio) alcancen un acuerdo económico sujeto a unas normas y a un sistema de seguimiento., [EN] The red-legged partridge (Alectoris rufa) has suffered in recent decades a notable regression of its populations as a result of several factors, among which the changes generated in agricultural practices will be highlighted. In this sense, the abandonment of the cultivation of some cereals, such as wheat (Triticum spp.), has generated in certain areas an even more marked decline in the populations of this species, mainly because their diet is based on wheat grains falling to the ground after harvesting with the harvester. In the municipal hunting ground of Mosqueruela (Teruel) there are two entities that make the commercial use. On the one hand, there is the town hall that, being the owner of the hunting ground, benefits from some hunting species (Capra pyrenaica) and roe deer (Capreolus capreolus), which generate a high economic benefit. On the other hand, there is the "Sociedad de Cazadores Virgen de la Estrella", which is responsible for the use of other species such as quail (Coturnix coturnix), the wood pigeon (Columba palumbus) or the red-legged partridge, paying an annual rent to the city council for such a service. On the other hand, in the municipality there are several farmers who carry out agricultural tasks for other farm owners or for their own plots, but who receive very low profit margins for their crops. In this sense, this group would be willing to grow more land (with certain products such as wheat), if given facilities or financial aid of any kind. In this context, the objective of the present work is to create an environmental payment system (PSA) with which to get environmental service plaintiffs (the council and the hunters' company) and bidders (farmers) in economic agreement subject to rules and a monitoring system.

[EN] BackgroundThe impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species.ResultsEuropean eel males treated with cold seawater (10 degrees C, T10) for 2weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others.ConclusionsCold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The

[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.

[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.

[EN] Maturation in captivity of European eel (Anguilla anguilla) requires long and costly hormonal treatments that often lead to asynchronic maturation between sexes. Therefore, optimization of sperm short-term storage methods and cryopreservation protocols can be a key factor for successful artificial fertilization. Two experiments were carried out to optimize the existing protocols. For the short-term storage experiment, sperm was diluted in P1 extender and then stored at different dilution ratios (1,9 and 1,49). The best outcome was then tested at different temperatures (4 and 20¿°C) and in constant agitation or still. In the cryopreservation experiments, large sperm volumes (cryotubes of 2 and 5¿ml), different cooling rates (freezing tubes 1 or 3¿cm above liquid nitrogen during 15 and 20¿min), and different extender compositions (methanol 10% was used as cryoprotectant, and complemented with FBS 20%, BSA 5% or egg yolk 5%) were tested. Sperm kinetic parameters were analyzed with a CASA-Mot system both in fresh and short- or long-term stored samples. In the short-term storage trial, sperm quality did not show significant differences in the first 24¿h after sperm collection between the different storage conditions tested. For longer time, 1:49 dilution ratio showed significantly better results than 1:9, and low temperature (4¿°C) was better for sperm preservation after 3¿days. Cryopreserved sperm samples showed good motility results when they were frozen in cryotubes of 2 and 5¿ml, with no significant differences compared to samples cryopreserved in lower volumes (straws of 0.5¿mL). Furthermore, the combination of methanol (10%) and egg yolk (5%) as freezing medium, induced significant higher post-thawing motility values (over 50%) than the control (methanol 10%), whereas the addition of FBS (20%) and BSA (5%) led to a significant reduction of the sperm motility. The establishment of these storage and cryopreservation protocols will be important for the improvemen

[EN] In the past three decades the European eel Anguilla anguilla experienced up to 99% decline in recruitment in some parts of its distribution range, thus breeding in captivity is nowadays considered key in order to save this species. With this in mind, obtaining high quality gametes is fundamental, as is the ongoing study of new hormonal treatments in order to improve current methods. Therefore, the aim of this research study was i) to assess the effect of two hormonal treatments (OVI, a recombinant alpha-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine) on the reproductive performance of European eel males, and, after choosing the best hormone, ii) to compare the effects of three doses in order to cut the costs of artificial maturation. Our results indicated that the type of hormone used (recombinant vs purified gonadotropins) significantly affected the progression of spermiation in European eel males, and that the recombinant hormone (OVI) produced better results in terms of sperm quantity and quality in most of the weeks of the treatment, remaining thus an effective treatment to induce spermiation in this species. On the other hand, in terms of the doses experiment, our results showed that from the lowest to the highest dose (0.25 to 1.5 IU/g fish) all the treatments were able to induce the whole spermiation process. However, a weekly dose of 1.5 IU/g fish of recombinant hormone (OVI) was necessary in order to provide a notable amount (volume and density) of high quality (motility and velocity) samples throughout the treatment. Finally, the economic analysis demonstrated that the recombinant hormone (OVI, 1.5 IU/g fish) had a greater profitability than the other treatments, making it possible to obtain high-quality sperm for a lower price. In this context, and considering the fact that in the first few weeks of any hormonal treatment there is no high-quality sperm production, long-term hormonal therapies are necessary in o

Tesis por compendio, [ES] En las últimas décadas, la anguila europea Anguilla anguila ha sufrido una disminución drástica de su población lo que ha llevado su inclusión como especie en peligro crítico en la lista roja UICN. Esta situación, junto con la gran importancia comercial de esta especie, evidencia la necesidad de tomar acciones como el desarrollo de la reproducción en cautividad y el control de la pesca. Una de las herramientas más interesantes para su uso en la biología de la conservación es la criopreservación de espermatozoides, que presenta varias ventajas para esta especie, incluyendo la sincronización de gametos, la selección de líneas genéticas o su uso para la creación de un criobanco. Sin embargo, el desarrollo de protocolos de criopreservación necesariamente requieren esperma de buena calidad. Además, se necesita un método preciso para evaluar la calidad del esperma tanto antes como después de la criopreservación. Sobre esta última cuestión, la motilidad de los espermatozoides de los peces se considera uno de los mejores biomarcadores para la evaluación de la calidad de los espermatozoides en los peces, y se puede estudiar de forma subjetiva u objetiva utilizando sistemas "computer assisted sperm analysis" (CASA-Mot). Primero, se realizó un experimento para evaluar la precisión y la exactitud de ambos métodos para estudiar la motilidad del esperma: el método subjetivo y la técnica objetiva que utiliza el sistema CASA-Mot. Además, se probó si el grado de experiencia de los técnicos en el caso del método subjetivo tiene un efecto en la precisión de la estimación de la motilidad y, por lo tanto, hay una influencia del personal del laboratorio en la evaluación de la motilidad del esperma. Aquí concluimos que tanto el método como la experiencia técnica eran factores clave para evaluar con precisión la motilidad del esperma en la anguila europea, por lo que se requiere el uso de CASA-Mot junto con material calificado para obtener resultados fehacientes. En segundo lugar, se, [CA] En les últimes dècades, l'anguila europea Anguilla anguila ha sofert una disminució dràstica de la seva població el que ha portat la seva inclusió com a espècie en perill crític en la llista vermella UICN. Aquesta situació, juntament amb la gran importància comercial d'aquesta espècie, evidencia la necessitat de prendre accions com el desenvolupament de la reproducció en captivitat i el control de la pesca. Una de les eines més interessants per al seu ús en la biologia de la conservació és la criopreservació d'espermatozoides, que presenta diversos avantatges per a aquesta espècie, incloent la sincronització de gàmetes, la selecció de línies genètiques o el seu ús per a la creació d'un criobanc. No obstant això, el desenvolupament de protocols de criopreservació necessàriament requereixen esperma de bona qualitat. A més, es necessita un mètode precís per avaluar la qualitat de l'esperma tant abans com després de la criopreservació. Sobre aquesta última qüestió, la motilitat dels espermatozoides dels peixos es considera un dels millors biomarcadors per a l'avaluació de la qualitat dels espermatozoides en els peixos, i es pot estudiar de forma subjectiva o objectiva utilitzant sistemes "computer assisted sperm analysis" (CASA-Mot). Primer, es va realitzar un experiment per avaluar la precisió i l'exactitud de tots dos mètodes per estudiar la motilitat de l'esperma: el mètode subjectiu i la tècnica objectiva que utilitza el sistema CASA-Mot. A més, es va provar si el grau d'experiència dels tècnics en el cas del mètode subjectiu té un efecte en la precisió de l'estimació de la motilitat i, per tant, hi ha una influència del personal del laboratori en l'avaluació de la motilitat del esperma. Vam concloure que tant el mètode com l'experiència tècnica eren factors clau per avaluar amb precisió la motilitat de l'esperma en l'anguila europea, de manera que es requereix l'ús de CASA-Mot juntament amb material qualificat per obtenir resultats fefaents. En segon lloc, es, [EN] In the last decades, the European eel Anguilla anguilla has suffered a drastic decrease in the recruitment in most areas of their distribution range, leading the species to be included as critically endangered in the IUCN list. This situation, together with the high commercial importance of the species, evidence the need of taking actions such as development of reproduction in captivity and control of fisheries based on the complexity of their life cycle. One of the most interesting tools for its use in conservation biology is the sperm cryopreservation, which presents several advantages for this species such as the synchronization of gametes, selection of genetic lines or cryobanking. However, the development of cryopreservation protocols necessarily requires good quality sperm, and it is also needed an accurate method to assess sperm quality both pre- and post-cryopreservation. On this last matter, fish sperm motility is considered one of the best quality biomarkers for sperm quality assessment in fish, and it can be evaluated subjectively or objectively using computer assisted sperm analysis (CASA-Mot) systems. First, an experiment was conducted to evaluate the precision and accuracy of both methods for assessing sperm motility: the subjective method and the objective technique using CASA-Mot system. Moreover, it was tested whether the degree of expertise of the technicians in the case of the subjective method, has an effect on the accuracy of the motility estimation, and therefore there is an influence of the laboratory staff on the sperm motility assessment. Here we concluded that both the method and the technician expertise were key factors in order to accurately assess sperm motility in European eel, so the use of CASA-Mot together with qualified stuff is required to obtain reliable results. Secondly, and alternative methods for European eel males maturation was evaluated by testing two different hormonal treatments: OVI, a recombinant ¿-choriogonadotrop

[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.

[EN] Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the afore-mentioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation. Crown Copyright (C) 2019 Published by Elsevier Inc. All rights reserved.

[EN] The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopreservation protocols for reproduction in captivity and cryobanking. In the last years, two research groups have developed their own protocols in Spain and Hungary with positive results, but difficult to compare. Here, a series of experiments were conducted to test the quality of thawed sperm after using both protocols, determining which of them produce the best results and aiming for standardization. The quality of thawed sperm was assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocols using a computer-assisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performed using flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in spermatozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryoprotectants (DMSO or methanol) that may induce epigenetic changes in the sperm DNA (cytosine methylation) and could affect the offspring, we conducted a luminometric methylation assay (LUMA) to study the DNA methylation levels induced by both protocols. In this work, all the above-mentioned parameters were analyzed in fresh and frozen-thawed sperm samples. Our results showed that thawed sperm samples from both protocols presented lower sperm motility and velocity, and lower percentage of live cells than those shown in fresh sperm samples. Furthermore, sperm samples from the methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoa than the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocol induced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol did not alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitable protocol that preserv

[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.

[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.

[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.

[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.

[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.

[EN] The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing from this media, and by testing the use of channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pH; in the quiescent stage was also induced. Intracellular pH (pH;) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pH; (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

[EN] Tetraodontidae (pufferfish) family members carry the smallest genomes among vertebrates, and these pocket-sized genomes have directly contributed to our understanding of the structure and evolution of higher animals. The grass pufferfish (Takifugu niphobles) could be considered a potential new model organism for comparative genomics and development due to the potential access to embryos, and availability of sequence data for two similar genomes: that of spotted green pufferfish (Tetraodon nigroviridis) and Fugu (Takifugu rubripes). In this study, we provide the first description of the normal embryonic development of T niphobles, by drawing comparisons with the closely related species cited above. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored at a constant temperature consistent with natural conditions. T. niphobles development was divided into seven periods of embryogenesis: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods; and stages subdividing these periods are defined based on morphological characteristics. The developmental stage series described in this study aims to provide the utilization of T niphobles as an experimental model organism for comparative developmental studies.

[EN] This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10 degrees C (first 4 weeks), 15 degrees C (next 3 weeks) and 20 degrees C (last 6 weeks); T15: 15 degrees C (first 4 weeks) and 20 degrees C (last 9 weeks); and T20: constant 20 degrees C for the duration of the experiment. At 10 degrees C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was >= 15 degrees C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11 beta HSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2).Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was >= 15 degrees C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration. (C) 2016 Elsevier Inc. All rights reserved.

[EN] The role of seminal plasma sodium and activation media sodium on sperm motility was examined by selectively removing the element from these two media, in European eel sperm. Sperm size (sperm head area) was also measured using an ASMA (Automated Sperm Morphometry Analyses) system, in the different conditions. Intracellular sodium [Na+](i) was quantitatively analyzed by first time in the spermatozoa from a marine fish species. Measurement of [Na+](i) was done before and after motility activation, by Flow Cytometry, using CoroNa Green AM as a dye. Sperm motility activation induced an increase in [Na+](i) from 96.72 mM in quiescent stage to 152.21 mM post-activation in seawater. A significant decrease in sperm head area was observed post activation in seawater. There was a notable reduction in sperm motility when sodium was removed from the seminal plasma, but not when it was removed from the activation media. Sodium removal was also linked to a significant reduction in sperm head area in comparison to the controls. Our results indicate that the presence of the ion Na+ in the seminal plasma (or in the extender medium) is necessary for the preservation of sperm motility in European eel, probably because it plays a role in maintaining an appropriate sperm cell volume in the quiescent stage of the spermatozoa. (C) 2016 Elsevier Inc All rights reserved.

[EN] Sperm from European eel males treated with hCG(rec), was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCI + EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+](i) fluorescence (and in [Na+](i) in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCI + EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+](i) levels did not increase at all. This result strongly suggests that increasing [Ca2+](i) is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+](i) concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm. (c) 2015 Elsevier Inc All rights reserved.

[EN] By first time, 2DE protein profile of European eel seminal plasma has been determined. 14 different proteins corresponding to 9 major families were identified in seminal plasma, through hormonal treatment. Some of them play a part in sperm maturation, including carbonic anhydrase which is responsible for modulating the pH of seminal plasma, and warm temperature acclimation protein, which may play an important role in the final maturation of this species, due to the warm temperature of their spawning ground (in the Sargasso Sea). Sperm samples were classified into three motility categories depending on the percentage of motile cells, I: 0-25%, II: 25-50% and 111: >50%. Different protein profiles were observed depending on the sperm motility categories, specifically, with the apolipoproteins and complement C3. Higher numbers of proteins from the apolipoprotein family were registered at lower motilities; whereas the complement C3-like family was higher in the samples with the highest percentage of motile cells. These results suggest that the proteins linked to the transportation of lipids (apolipoprotein) and to the immune system (complement C3) may carry out their functions at different stages of spermatogenesis. Using SDS-PAGE analysis, 13 bands were identified, most of which migrated between 20 to 60 kDa. In the last weeks of treatment significant increases were observed in the percentage of motile spermatozoa, curvilinear velocity and beat cross frequency. This improvement in sperm quality coincided with a higher amount of proteins located at 19 KDa, therefore, this protein could be involved in sperm motility of the European eel. (C) 2016 Published by Elsevier Inc.

[EN] This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic add (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of alpha-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species. (C) 2014 Elsevier Inc All rights reserved.

[EN] There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90 s), the thermal regimen (i.e. 10 degrees C (T10) or 15 degrees C (T15) and up to 20 degrees C, or constant at 20 degrees C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30 s is recommended as the standard time for motility acquisition. When eels were kept at 20 degrees C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8 +/- 1.3%; mean +/- s.e.m.) and more stable

[EN] Positive effects of probiotics on fish reproduction have been reported in several species. In the present study, 40 male European eels were weekly treated with recombinant hCG for 9 weeks and with three different concentrations (10(3), 10(5), and 10(6) CFU/mL) of probiotic Lactobacillus rhamnosus IMC 501 (Sinbyotec, Italy). The probiotics were daily added to the water from the sixth week of the hCG treatment. Males from the treated and control groups were sacrificed after 1, 2, and 3 weeks of probiotic treatment (seventh ninth weeks of hCG treatment); at this point, sperm and testis samples were also collected. Sperm volume was estimated, and motility was analyzed by computer-assisted sperm analysis software. Alternations in transcription of specific genes involved in reproductive process such as activin, androgen receptors alpha and beta (ar alpha and ar beta), progesterone receptor 1 (pr1), bone morphogenetic protein 15 (bmp15), and FSH receptor (fshr) were analyzed in the testis. After 2 weeks of probiotic treatment, sperm production and sperm motility parameters (percentage of motile cells and percentage of straight-swimming spermatozoa) were increased in the European eel treated with 105 CFU/mL compared to controls or to the other probiotic doses. These changes were associated with increases in messenger RNA expression of activin, ar alpha, ar beta, pr1, and fshr. Conversely, after 3 weeks, activin and pr1 expression decreased. No significant changes were observed on bmp15 expression throughout the duration of the treatment with 10(5) CFU/mL dose. The lowest and highest probiotic dose (10(3) and 10(6) CFU/mL, respectively) inhibited the transcription of all genes along all the experiment, except for ar alpha and ar beta after 1 week of probiotic treatment when compared to controls. The changes observed by transcriptomic analysis and the sperm parameters suggest that a treatment with L rhamnosus at 10(5) CFU/mL for 2 weeks could improve spermatogenesis proc

[EN] Activation at fertilization of the vertebrate egg is triggered by Ca2+ waves. Recent studies suggest the phospholipase C zeta (PLC zeta), a sperm-specific protein, triggers egg activation by an 1P3-mediated Ca2+ release and allow Ca2+ waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLC zeta (PLC zeta 1). It is 1521bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLC zeta orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plc zeta was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plc zeta 1 expression in the pituitary gland. Testis plc zeta 1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLC zeta inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes. (C) 2015 Elsevier Inc. All rights reserved.

[EN] The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy. (C) 2014 Elsevier Inc. All rights reserved.

[EN] The objective of this study was evaluating the effects on the growth rate and flesh quality of separating larvae according to their larval width in cultured gilthead seabream (Sparus aurata, L.). Progeny from two broodstocks (A and B) were divided into two groups according to larval width: heads (being the larger specimens) and tails. After 18months both groups were analysed to evaluate growth, carcass traits and proximal composition. The head specimens reached a larger size and showed a greater level of well-being and degree of nourishment. Total body and fillet composition in general were found to be as expected for this species. Some differences were found in total body composition between head and tail specimens in both batches, and in the fillet composition in batch B (higher fat and lower moisture content in the head specimens). Sensory analyses were carried out using untrained panelists, who were unable to distinguish between the head and tail samples in batch A, whereas differences were noticeable in batch B. Samples from the head group were judged to be tastier and juicier, a consequence of their higher fat content. Hence, fish separation according to larval width is an effective tool to separate progeny into slow and fast growing groups, whereas the total body and fillet analyses and a sensory test ensure that the selection does not generate negative effects on the product quality.

[EN] This research investigated the regulation of aromatase and androgen receptor gene expression in the brain–pituitary– gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa-cyp19a1) and partial androgen receptor a and b (aa-ara and aa-arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa-arb was expressed at higher levels than aa-ara in the pituitary and gonad in both sexes, suggesting aa-arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa-ara and an increase in aa-cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa-arb and aa-cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa-arb and an increase in aa-cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa-cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex-dependent differences in the regulation of the expression of aa-ara, aa-arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation

[EN] Little is known about the role of fat and fatty acids in European eel spermatogenesis. The aim of this research was to study the changes in fat content and to carry out a quantitative analysis of the fatty acid composition of the muscle, liver and gonad of European male eels during hormonally induced sexual maturation. Three different thermal regimes were used (two variable: T10 and T15; and one constant: T20) to replicate the changes in temperature that these fish experience during their transoceanic reproductive migration. Spermatogenesis was reached earlier in treatment T20, suggesting that spermatogenesis in the European eel is closely regulated by water temperature. Although eels lose body mass due to the period of fasting that accompanies gonadal growth, no significant changes were found in the fat and fatty acid content of the muscle during the experimental period. With regard to the liver, the levels of palmitic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids increased significantly at the start of the spermiation process in all the thermal treatments. In the testis, levels of EPA, arachidonic acid (ARA) and DHA remained constant during the maturation process whereas the levels of the rest of the fatty acids decreased significantly. The stability of the ARA and EPA levels in the testis may have a physiological significance, whereas the stability of the DHA levels may have a structural significance. The results suggest that the progression of spermiation is influenced by water temperature and demonstrate the importance of EPA, ARA and DHA in European eel reproduction. This study demonstrates that complementary research focusing on the lipid composition of commercial diets could improve the sperm quality of this species.