Development of innovative methods for induction of European eel (Anguilla anguilla) spermatogenesis

Tesis por compendio[ES] Resumen Como pez de gran valor económico, procedente de una de las líneas de teleósteos más antiguas, con un ciclo de vida misterioso, un potencial de acuicultura excepcional, y con importancia cultural y actividades de pesca en casi todos los países de Europa, la anguila europea posee un enorme valor socioeconómico. Este valor se suma a la desgraciada situación actual en peligro crítico de población natural de anguilas europeas. Como el ciclo de vida de la anguila aún no se ha conseguido cerrar en cautiverio, si la especie se extingue en la naturaleza, no seremos capaces de recuperarla. El cierre del ciclo de vida de la anguila europea ha sido, por lo tanto, el objetivo final de varios estudios. Sin embargo, a pesar de una investigación científica sustancial, desde la década de 1930, varios aspectos de la maduración de la anguila, como el mecanismo que bloquea la maduración de la anguila en la etapa prepúber en cautiverio, aún no se conocen bien. Por lo tanto, es necesario ampliar nuestro conocimiento sobre la reproducción de la anguila para inducir mejores hipótesis y lograr un progreso sustancial. Para profundizar en este campo, esta tesis se realizó con el objetivo específico de desarrollar métodos innovadores para la inducción de la maduración de la anguila y aumentar el conjunto de conocimientos sobre los procesos europeos de maduración de la anguila. Los procedimientos hormonales utilizados actualmente para la maduración sexual de la anguila artificial probablemente no induzcan el proceso natural de maduración. Por lo tanto, esta tesis ha evaluado el potencial de las hormonas recombinantes específicas de la anguila para inducir un proceso de maduración más natural. Este estudio específico mostró que la espermatogénesis completa y la espermiación se pueden inducir con gonadotropinas específicas de anguila recombinante; sin embargo, la calidad del gameto resultante es aún inferior a los resultados de los protocolos establecidos. Sin embargo, la utilización de hormonas recombinantes tiene un gran potencial para futuras implementaciones. Además, el experimento de gonadotropina recombinante ha generado nuevos detalles sobre el efecto de las gonadotropinas homólogas en el eje BPG de las anguilas europeas. Trabajos previos han llevado a la hipótesis de que un tratamiento térmico adecuado puede reducir o reemplazar parcialmente los tratamientos hormonales estándar para la maduración sexual de la anguila europea, o puede mejorar la calidad y / o cantidad de gametos. En esta tesis, se probó el efecto de varios regímenes térmicos en el eje BPG de machos de anguila europeos prepúberes, sin administración de hormonas. Los resultados muestran claramente que un tratamiento de agua de mar fría durante 2 semanas (10 ° C) afecta el eje BPG de los machos de anguila europeas. Los resultados específicos incluyeron un aumento en la sincronización de espermatogonias, niveles elevados de testosterona y 11-ketotestosterona en plasma, agrupamiento de muestras de transcriptomas del eje BPG del grupo tratado con agua de mar fría y posiblemente niveles aumentados de la proteína subunidad ß de la hormona luteinizante de la hipófisis. Los genes transcritos diferencialmente incluyeron varios genes, procesos y vías interesantes, que parecen estar involucrados en la maduración "natural" temprana de la anguila y que pueden ser biomarcadores adecuados para las distintas etapas de este proceso. Para un análisis adecuado de los datos transcriptómicos, se creó un transcriptoma de anguila europea de novo. Se demostró que este transcriptoma de novo posee una superior integridad al genoma de anguila europea disponible y, por lo tanto, es una herramienta útil para el análisis adicional de genes específicos. Un análisis de este transcriptoma reveló un gran número de pares de genes parálogos, que mostraron una baja divergencia entre secuencias sinónimas. Entre las hipótesis potenciales sobre e[CA] Com a espècie de renom culinari que pertany a un dels llinatges teleostis més antics, amb un cicle vital misteriós, un potencial d'aqüicultura excepcional, i una tradició pesquera a gairebé tots els països d'Europa, l'anguila europea posseeix un enorme valor socioeconòmic. No obstant això, aquest valor només fa que augmentar la preocupació de la seva població, que actualment es troba catalogada com "en perill crític d'extinció". Atès que el cicle de vida de les anguiles encara no ha estat tancat en captivitat, l'espècie no serà salvable en el cas que s'extingeixi en estat natural, per la qual cosa tancar el cicle de vida d'aquesta espècie ha estat l'objectiu final de diversos grups d'investigació durant els últims anys.. No obstant això, i malgrat la investigació científica de qualitat duta a terme des de la dècada de 1930, encara hi han diversos aspectes de la maduració de les anguiles -com el mecanisme que bloqueja la maduració sexual de l'anguila a l'etapa pre-puberal en captivitat- que son poc coneguts en l'actualitat. Per tal d'ampliar els coneixements sobre la reproducció de les anguiles i aconseguir un progrés substancial, aquesta tesi es va dur a terme amb l'objectiu específic de desenvolupar mètodes innovadors per a la inducció de la maduració de l'anguila europea, a més de afegir-hi el coneixement en els processos de maduració bàsics d'aquesta espècie. Els procediments hormonals utilitzats actualment per a la maduració artificial de l'anguila europea no acaben d'induir el procés de maduració natural tal i com probablement es dóna a la natura. Doncs, en primer lloc, aquesta tesi va avaluar el potencial d'hormones recombinants específiques d'anguila europea per induir un procés de maduració molt més natural. Aquest estudi específic va mostrar que mitjançant estes gonadotropines específiques d'anguila europea és possible induir l'espermatogènesi i l'espermiació completes. Tot i que els resultats van mostrar que la qualitat dels gamets va ser inferior als resultats que generen els protocols establerts fins ara amb un altre tipus d'hormones (generalment d'origen humà), la utilització d'hormones recombinants específiques es presenta amb un gran potencial per a la seva implementació futura en la inducció de la maduració sexual de l'anguila europea, ja que l'estudi va generar noves idees sobre l'efecte de les gonadotropines l'eix BPG de l'anguila europea. En segon lloc, i treballant amb la hipòtesi que un tractament tèrmic adequat pot reduir o substituir parcialment els tractaments hormonals estàndards per a la maduració sexual de l'anguila europea, en aquesta tesi es va provar l'efecte de diversos règims tèrmics (sense administració d'hormones) en l'eix BPG dels mascles europeus pre-puberals amb l'objectiu de millorar la qualitat i / o quantitat dels gamets. Els resultats mostraren clarament que un tractament d'aigua de mar de 2 setmanes a baixa temperatura (10 °C) va afectar l'eix BPG dels mascles europeus d'anguila. Resultats més específics van mostrar un augment de la sincronització de les espermatogonies, elevats nivells plasmàtics de testosterona i 11-ketotestosterona, una agrupació de mostres de transcriptoma de l'eix BPG del grup tractat amb aigua de mar freda i, possiblement, un augment dels nivells de la proteïna de la subunitat ß de la hormona luteinitzant de la hipofisi. Els gens transcrits diferencials van al·ludir a diversos gens, processos i vies interessants, que semblen estar implicats en la maduració inicial de l'anguila "natural" i podrien resultar biomarcadors adequats per a les etapes d'aquest procés. No obstant això, es necessiten estudis addicionals per avaluar el potencial dels biomarcadors d'aquests gens i, de manera complementària, comprovar si un pre-tractament d'aigua de mar freda pot millorar la resposta de les anguiles europees a un tractament hormonal artificial, com suggereixen els resultats. Finalment, amb l'objectiu[EN] As an expensive fish from one of the most ancient teleost lineages, with a mysterious life cycle, exceptional aquaculture potential, and cultural associations and fishing activity in almost every country in Europe, the European eel possess huge socioeconomic value. This value only adds to the misfortune of the current critically endangered state of the wild European eel population. As the eel lifecycle has not yet been closed in captivity, the species will not be salvable if it went extinct in the wild. Closing the life-cycle of the European eel has thus been the ultimate objective of several studies. However, despite the substantial scientific investigation, since the 1930s, several aspects of eel maturation, such as the mechanism which blocks eel sexual maturation at the pre-pubertal stage in captivity, is still poorly understood. Therefore, it is necessary to broaden our knowledge of eel reproduction to induce better hypotheses and therethrough achieve substantial progress. In order to further this field, this thesis was conducted with the specific objective of developing innovative methods for induction of eel maturation and add to the pool of knowledge of European eel maturation processes. The hormonal procedures currently used for artificial eel sexual maturation are probably not inducing the natural maturation process. Therefore, this thesis has evaluated the potential of eel specific recombinant hormones to induce a more natural maturation process. This specific study showed that full spermatogenesis and spermiation can be induced with recombinant eel specific gonadotropins; however, the resulting gamete quality is still inferior to the results of established protocols. Nevertheless, the utilization of recombinant hormones holds a large potential for future implementation. Furthermore, the recombinant gonadotropin experiment has generated novel insights into the effect of homologous gonadotropins on the BPG axis of European eels. Previous work has led to the hypothesis that the right thermal environmental treatment may reduce or partially replace the standard hormonal treatments for sexual maturation of European eel, or may improve gamete quality and/or quantity. In this thesis, the effect of various thermal regimes was tested on the BPG axis of pre-pubertal European eel males, without administration of hormones. The results clearly show that a 2 week cold (10 °C) seawater treatment effects the BPG-axis of European eel males. Specific results included an increase in the synchronization of spermatogonial cells, elevated testosterone and 11-ketotestosterone plasma levels, clustering of BPG-axis transcriptome samples from the cold seawater treated group and possibly increased levels of pituitary luteinizing hormone ß-subunit protein. Differentially transcribed genes alluded to several interesting genes, processes, and pathways, which appears to be involved in early "natural" eel maturation and may prove to be suitable biomarkers for the stages of this process. In order for proper analysis of the transcriptomic data, a de novo European eel transcriptome was assembled. This de novo transcriptome was proven to have superior completeness to the available European eel genome and is thus a useful tool for further analysis of specific genes. An analysis of this transcriptome revealed a large number of paralog gene pairs, which showed low synonymous sequence divergence. Among the potential hypothesis regarding the origin of these paralog gene pairs, the hypothesis of a 4R whole genome duplication is among the most parsimonious. Several of these duplicated genes were involved in reproduction and the onset of puberty. Regardless of the origin, further analysis of these genes may reveal eel specific adaptations, which could help to better understand the exceptional reproductive system of eels.Rozenfeld, C. (2019). Development of innovative methods for induction of European eel (Anguilla anguilla) spermatogenesis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125697TESISCompendi

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). Pakistan J Biol Sci 9:2141–2144. https://doi.org/10.3923/pjbs.2006.2141.2144Cabrita E, Martínez-Páramo S, Gavaia PJ, Riesco MF, Valcarce DG, Sarasquete C, Herráez MP, Robles V (2014) Factors enhancing fish sperm quality and emerging tools for sperm analysis. Aquaculture 432:389–401. https://doi.org/10.1016/j.aquaculture.2014.04.034Castellini C, Dal Bosco A, Ruggeri S, Collodel G (2011) What is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis? Fertil Steril 96(1):24–27Fauvel C, Suquet M, Cosson J (2010) Evaluation of fish sperm quality. J Appl Ichthyol 26:636–643. https://doi.org/10.1111/j.1439-0426.2010.01529.xGage MJG, Macfarlane CP, Yeates S, Ward RG, Searle JB, Parker GA (2004) Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. 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

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

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

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

Abundance of specific mRNA transcripts impacts hatching success in European eel, Anguilla anguilla L

International audienceMaternal mRNA governs early embryonic development in fish and variation in abundance of maternal transcripts may contribute to variation in embryonic survival and hatch success in European eel, Anguilla anguilla. Previous studies have shown that quantities of the maternal gene products β-tubulin, insulin-like growth factor 2 (igf2), nucleoplasmin (npm2), prohibitin 2 (phb2), phosphatidylinositol glycan biosynthesis class F protein 5 (pigf5), and carnitine O-palmitoyltransferase liver isoform-like 1 (cpt1) are associated with embryonic developmental competence in other teleosts. Here, the relations between relative mRNA abundance of these genes in eggs and/or embryos and egg quality, was studied and analyzed. We compared egg quality of the two groups: i) batches with hatching and ii) batches with no hatching. Results showed no significant differences in relative mRNA abundance between the hatch and no hatching groups for any of the selected genes at 0, 2.5, and 5 HPF. However, at 30 HPF the hatch group showed significantly higher abundance of cpt1a, cpt1b, β-tubulin, phb2, and pigf5 transcripts than the no hatch group. Therefore, these results indicate that up-regulation of the transcription of these genes in European eel after the mid-blastula transition, may be needed to sustain embryonic development and hatching success

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males

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