Improvement of techniques for sperm evaluation and cryobanking in European eel

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 evaluaron métodos alternativos para la maduración de los machos de anguila europeos probando dos tratamientos hormonales diferentes: OVI, una gonadotropina recombinante; y VET, una gonadotropina purificada a partir de orina femenina. Después de elegir el mejor tratamiento hormonal de los dos, se evaluó el efecto de tres dosis diferentes con el objetivo de obtener el mayor rendimiento al menor coste. Los resultados de este experimento apuntaron a OVI como el mejor tratamiento hormonal en una dosis semanal de 1.5 UI/g de pez, que proporciona la mayor rentabilidad, obteniendo esperma de alta calidad a menor precio. En un tercer experimento, y utilizando los conocimientos adquiridos en los dos primeros experimentos, se realizaron una serie de experimentos para estandarizar los protocolos de criopreservación de esperma de anguila europea disponibles en ese momento (utilizando DMSO o metanol como crioprotector). Los resultados apuntaron al protocolo que utiliza el metanol como el mejor de ellos dos en términos de motilidad, velocidad y viabilidad de los espermatozoides y la preservación de la integridad del ADN. Siguiendo este último método estandarizado, se realizó un cuarto experimento con el objetivo de mejorar el protocolo en términos de volumen (volúmenes de esperma más grandes) y de calidad espermática. Además, se desarrolló un protocolo simple de almacenamiento a corto plazo para complementar las opciones de preservar el esperma durante diferentes períodos de tiempo. De todas las condiciones de almacenamiento probadas, las diluciones 1/50 a 4 ºC mostraron los mejores resultados, manteniendo la motilidad en comparación con el control durante 3 días, y manteniendo cierta motilidad espermática (12%) después de 7 días. A partir del experimento de criopreservación, fue posible aumentar los volúmenes a 2 y 5 mL sin perder la calidad del esperma en comparación con volúmenes más pequeños, y mejorando las mot[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 van avaluar mètodes alternatius per a la maduració dels mascles d'anguila europeus provant dos tractaments hormonals diferents: OVI, un gonadotropina recombinant; i VET, un gonadotropina purificada a partir d'orina femenina. Després de triar el millor tractament hormonal dels dos, es va avaluar l'efecte de tres dosis diferents amb l'objectiu d'obtenir el major rendiment al menor cost. Els resultats d'aquest experiment van apuntar a OVI com el millor tractament hormonal en una dosi setmanal de 1.5 UI/g de peix, que proporciona la major rendibilitat, obtenint esperma d'alta qualitat a un preu millor. En un tercer experiment, i utilitzant els coneixements adquirits en els dos primers experiments, es van realitzar una sèrie d'experiments per estandarditzar els protocols de criopreservació d'esperma d'anguila europea disponibles en aquest moment (utilitzant DMSO o metanol com crioprotector). Els resultats van apuntar al protocol que utilitza el metanol com el millor d'ells dos en termes de motilitat, velocitat i viabilitat dels espermatozoides i la preservació de la integritat de l'ADN. Seguint aquest últim mètode estandarditzat, es va realitzar un quart experiment amb l'objectiu de millorar el protocol en termes de volum (volums d'esperma més grans) i de qualitat espermàtica. A més, es va desenvolupar un protocol simple d'emmagatzematge a curt termini per complementar les opcions de preservar l'esperma durant diferents períodes de temps. De totes les condicions d'emmagatzematge provades, les dilucions 1/50 a 4ºC van mostrar els millors resultats, mantenint la motilitat en comparació amb el control durant 3 dies, i mantenint certa motilitat espermàtica (12%) després de 7 dies. A partir de l'experiment de criopreservació, va ser possible augmentar els volums a 2 i 5 ml sense perdre la qualitat de l'esperma en comparació amb volums més petits.[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 ¿-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine. After choosing the best hormonal treatment, the effect of three different doses was evaluated aiming for best performance and lowest cost on the treatment. The results of this experiment pointed at OVI as the best hormonal treatment in terms on sperm quantity and quality in most of the weeks of treatment, and at a weekly dose of 1.5 IU/g fish, which also provide the greatest profitability, obtaining high quality sperm at a lower price. In a third experiment, and using the knowledge acquired in the two first experiments (using the OVI hormonal treatment and CASA-Mot to assess sperm quality), a series of experiments were conducted to standardize the European eel sperm cryopreservation protocols available at the moment (using DMSO or methanol as cryoprotectant). The results indicated that the protocol using methanol was the best of them two in terms of sperm motility and velocity, sperm viability and preservation of DNA integrity. Following this last standardized method, a fourth experiment was conducted aiming for improvement of the protocol in terms of volume (larger volumes) and sperm quality outcome. Moreover, a simple protocol for short-term storage was developed to complement the options to preserve sperm for different time periods. Of all the tested storing conditions, 1/50 dilutions at 4 ºC showed the best results, maintaining the motility compared to control for 3 days, and some sperm motility (12%) was still observed after 7 days. From the cryopreservation experiment, it was possible to scale up the cryopreserved volumes to 2 and 5 mL without losing sperm quality compared to lower volumes. Moreover, the protocol was further improved by supplementing the protocol with egg yolk as an additive, obtaining the highest cryopreserved sperm motilities (over 50%) ever reported in European eel.Herranz Jusdado, JG. (2019). Improvement of techniques for sperm evaluation and cryobanking in European eel [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130846TESISCompendi

Subjective and objective assessment of fish sperm motility: when the technique and technicians matter

[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, C.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2018). Subjective and objective assessment of fish sperm motility: when the technique and technicians matter. Fish Physiology and Biochemistry. 1-11. https://doi.org/10.1007/s10695-018-0505-1S111Boryshpolets S, Kowalski RK, Dietrich GJ, Dzyuba B, Ciereszko A (2013) Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters. Theriogenology 80:758–765. https://doi.org/10.1016/j.theriogenology.2013.06.019Bozkurt Y, Secer S (2006) Relationship between spermatozoa motility, egg size, fecundity and fertilization success in brown trout (Salmo trutta fario). 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Curr Biol 14:44–47. https://doi.org/10.1016/S0960-9822(03)00939-4Gallego V, Asturiano JF (2018) Sperm motility in fish: technical applications and perspectives through computer-aided sperm analysis (CASA) systems. Reprod Fertil Dev ( http://www.publish.csiro.au/RD/justaccepted/RD17460 )Gallego V, Carneiro PCF, Mazzeo I, Vílchez MC, Peñaranda DS, Soler C, Pérez L, Asturiano JF (2013a) Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology 79:1034–1040. https://doi.org/10.1016/j.theriogenology.2013.01.019Gallego V, Cavalcante SS, Fujimoto RY, Carneiro PCF, Azevedo HC, Maria AN (2017) Fish sperm subpopulations: changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum). Theriogenology 87:16–24. https://doi.org/10.1016/j.theriogenology.2016.08.001Gallego V, Pérez L, Asturiano JF, Yoshida M (2013b) Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles). Aquaculture 416–417:238–243. https://doi.org/10.1016/j.aquaculture.2013.08.035Gasparini C, Simmons LW, Beveridge M, Evans JP (2010) Sperm swimming velocity predicts competitive fertilization success in the green swordtail Xiphophorus helleri. PLoS One 5:e12146. https://doi.org/10.1371/journal.pone.0012146Hala DN, VanLook K, Holt WV, Jobling S (2009) Validation of a method for measuring sperm quality and quantity in reproductive toxicity tests with pair-breeding male fathead minnows (Pimephales promelas). ILAR J 50:e1–e10Kime DE, VanLook KJW, McAllister BG et al (2001) Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comp Biochem Physiol - C Toxicol Pharmacol 130:425–433. https://doi.org/10.1016/S1532-0456(01)00270-8Komatireddy R r, Madishetti R (2017) Coefficient of variation assessment for seminal traits evaluated by computer assisted semen analysis (CASA). Int J Sci Environ Technol 5:3452–3456Liu QH, Li J, Xiao ZZ, Ding FH, Yu DD, Xu XZ (2007) Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream (Pagrus major). Aquaculture 263:20–25. https://doi.org/10.1016/j.aquaculture.2006.11.017McAuliffe RE (2015) Coefficient of variation. In: Wiley encyclopedia of management. John Wiley & Sons, Ltd, Chichester, pp 1–1Peñaranda DS, Pérez L, Gallego V, Barrera R, Jover M, Asturiano JF (2010) European eel sperm diluent for short-term storage. Reprod Domest Anim 45:407–415. https://doi.org/10.1111/j.1439-0531.2008.01206.xPepper-Yowell AR (2011) The use of computer assisted semen analysis to predict fertility in Holstein bulls. Colorado State University. Doctoral ThesisPérez L, Asturiano JF, Tomás A et al (2000) Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. J Fish Biol 57:1488–1504. https://doi.org/10.1006/jfbi.2000.1411Reicks DL, Center SV, St Peter MN (2012) Capturing value of CASA-Mot systemsRosenthal H, Asturiano JF, Linhart O, Horváth Á (2010) On the biology of fish gametes: summary and recommendations of the Second International Workshop, Valencia, Spain, 2009. J Appl Ichthyol 26:621–622. https://doi.org/10.1111/j.1439-0426.2010.01550.xRudolfsen G, Figenschou L, Folstad I, Kleven O (2008) Sperm velocity influence paternity in the Atlantic cod (Gadus morhua L.). Aquac Res 39:212–216. https://doi.org/10.1111/j.1365-2109.2007.01863.xRurangwa E, Kime DE, Ollevier F, Nash JP (2004) The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture 234:1–28. https://doi.org/10.1016/j.aquaculture.2003.12.006Verstegen J, Iguer-Ouada M, Onclin K (2002) Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 57:149–179. https://doi.org/10.1016/S0093-691X(01)00664-1Walker JS, Winet H, Freund M (1982) A comparison of subjective and objective sperm motility evaluation. J Androl 3:184–192. https://doi.org/10.1002/j.1939-4640.1982.tb00667.xWorld Health Organization (2010). WHO laboratory manual for the examination and processing of human semen (5th edition). Printed in Switzerland (286 pp.

Recombinant vs purified mammal gonadotropins as maturation hormonal treatments of European eel males

[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 order to lessen the cost of high-quality European eel sperm.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 (ETN IMPRESS). VG has a postdoc grant from the UPV (PAID-10-16).Herranz-Jusdado, JG.; Rozenfeld, C.; Morini, M.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Gallego Albiach, V. (2019). Recombinant vs purified mammal gonadotropins as maturation hormonal treatments of European eel males. Aquaculture. 501:527-536. https://doi.org/10.1016/j.aquaculture.2018.12.015S52753650

European eel sperm storage: optimization of short-term protocols and cryopreservation of large volumes

[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 improvement of European eel artificial reproduction programs.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement N° 642893 (IMPRESS), including the JGHJ and CR predoctoral contracts. VG has a postdoc grant from the UPV (PAID-10-16).Herranz-Jusdado, JG.; Gallego Albiach, V.; Rozenfeld, C.; Morini, M.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2019). European eel sperm storage: optimization of short-term protocols and cryopreservation of large volumes. Aquaculture. 506:42-50. https://doi.org/10.1016/j.aquaculture.2019.03.019S425050

Eel sperm cryopreservation: an overview

[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 University as well as the EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union and the European Social Fund.Herranz-Jusdado, JG.; Gallego Albiach, V.; Morini, M.; Rozenfeld, C.; Pérez Igualada, LM.; Müller, T.; Horváth, Á.... (2019). Eel sperm cryopreservation: an overview. Theriogenology. 133:210-215. https://doi.org/10.1016/j.theriogenology.2019.03.033S21021513

CD38 Defines a Subset of B Cells in Rainbow Trout Kidney With High IgM Secreting Capacities

Funding Information: This work was supported by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM) and by the Comunidad de Madrid (grant 2016-T1/BIO-1672).Peer reviewedPublisher PD

Handling and treatment of male European eels (Anguilla anguilla) for hormonal maturation and sperm cryopreservation

[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/FEKUTHerranz-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/10.3791/56835S16131e5683

Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and Siberian sturgeon (Acipenser baerii)

[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 Siberian sturgeon (Acipenser baerii). Aquaculture. 502:223-231. https://doi.org/10.1016/j.aquaculture.2018.12.001S22323150

De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages

[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 also included the predoctoral contracts of CR and JGHJ. VG has a postdoctoral grant from the Spanish Ministry of Science, Innovation and Universities (MICIU; Programa Juan de la Cierva-Incorporacion; IJCI-2017-34200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Rozenfeld, C.; Blanca Postigo, JM.; Gallego Albiach, V.; García-Carpintero, V.; Herranz-Jusdado, JG.; Pérez Igualada, LM.; Asturiano, JF.... (2019). De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages. 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Comparison of European eel sperm cryopreservation protocols with standardization as a target

[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 preserves better the motility and genetic qualities of the European eel sperm.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 predoctoral contracts, the French CRB Anim project "Investissements d'avenir", ANR-11-INBS-0003, as well as the Hungarian EFOP 3.6.3.-VEKOP 16.-2017-00008 project, and by the Higher Education Institutional Excellence Program (1783-3/2018/FEKUTSRAT) awarded by the Ministry of Human Capacities within the framework of water related researches of Szent Istvan University. MM, EK, TK and AH were granted with Short Term Scientific Missions by the COST Office (COST Action FA1205: AQUAGAMETE).Herranz-Jusdado, JG.; Gallego Albiach, V.; Morini, M.; Rozenfeld, C.; Pérez Igualada, LM.; Kása, E.; Kollár, T.... (2019). Comparison of European eel sperm cryopreservation protocols with standardization as a target. Aquaculture. 498:539-544. https://doi.org/10.1016/j.aquaculture.2018.09.00653954449