Study of the effects of thermal regime and alternative hormonal treatments on the reproductive performance of European eel males (Anguilla anguilla) during induced sexual maturation

[EN] Since 1960, the European eel (Anguilla anguilla) has suffered a dramatic reduction in natural stocks. Breeding in captivity is considered an alternative, but obtaining high quality sperm seems basic on this regard. The main objective of this study was to assess the effects of three thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and three hormonal treatments with different hormones (hCG, hCGrec and PSMG) on the induction of maturation in European eel males. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced, and perhaps closely regulated, by water temperature. T20 demonstrated the best results in all the sperm parameters (volume, density, motility, kinetic features, etc.) throughout most weeks of treatment, becoming a reliable and productive method for inducing spermiation in this species. In the case of hormonal treatments, the onset and progression of spermiation in European eel males were influenced by the hormone used. In this respect, hCGrec produced the best results in all the sperm parameters including volume, density, motility, kinetic features, etc., throughout most weeks of treatment, thus becoming an effective alternative treatment to the standard hCG treatment used to induce spermiation in eel species. Moreover, hCGrec gave rise to the best economical profitability, making it possible to obtain good quality sperm samples at a lower price than by using the other two hormonal treatments. © 2012 Elsevier B.V. All rights reserved Guardar / Salir Siguiente >Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL) and Generalitat Valenciana (ACOMP/2012/086). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez have predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. Paulo C.F. Carneiro had post-doc grants from PAC-EMBRAPA and UPV PAID Programme (PAID-02-11). David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I).Gallego Albiach, V.; Mazzeo, I.; Vilchez Olivencia, MC.; Peñaranda, D.; Carneiro, PCF.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2012). Study of the effects of thermal regime and alternative hormonal treatments on the reproductive performance of European eel males (Anguilla anguilla) during induced sexual maturation. Aquaculture. 354:7-16. https://doi.org/10.1016/j.aquaculture.2012.04.041S71635

Hormonal induction of gonadal maturation and spawning in European eel females Anguilla Anguilla L., 1758: Morphological changes and oocyte development

The present study assesses different hormonal treatments for the induction of gonadal maturation and spawning in female wild European eels Anguilla anguilla L., 1758. One of the groups received weekly injections of salmon pituitary extract and human chorionic gonadotropin, while a second group received the same treatment together with pymozide. Although previous studies had reported spontaneous release of oocytes, in the present study, during the final phases of oocyte development, a dose of 17.20β-dihydroxy-4-pregnen-3-one (17.20βP) was used to induce the first controlled spawning in A. anguilla. After 12-14 weeks of treatment, 5 spawns were obtained. Treatments also caused morphological changes, such as progressively darker coloration and increase in eye diameter.El objetivo principal del presente estudio fue la evaluación de distintos tratamientos hormonales para la inducción de la maduración gonadal y la puesta en hembras salvajes de anguila europea Anguilla anguilla L., 1758.Un grupo recibió inyecciones semanales de extracto de hipófisis de salmón y gonadotropina coriónica humana, mientras que un segundo grupo recibió además pymozide. Aunque estudios previos habían logrado liberación espontánea de oocitos, en el presente estudio se empleó una dosis de 17,20 β-dihidroxi-4-pregnen-3-ona (17,20 βP) durante las fases finales del desarrollo oocitario para inducir de forma controlada, por primera vez en la anguila europea, la ovulación y puesta. Después de 12-14 semanas de tratamiento se obtuvieron 5 puestas. Los tratamientos también causaron cambios morfológicos, como el oscurecimiento de la coloración o el aumento del diámetro ocular.Instituto Español de Oceanografí

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

[EN] The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10x was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the camera's highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species. (C) 2013 Elsevier Inc. All rights reserved.This study was funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology," grant agreement 245257 (Pro-Eel), and Generalitat Valenciana (ACOMP/2011/229). D.S.P. and P.C.F.C. have postdoctoral grants from UPV (CE-01-10) and PAC-EMBRAPA, respectively. I.M. and V.G. have predoctoral grants from Generalitat Valenciana and Spanish MICINN, respectively. The authors thank the Proiser R&D, S.L. team, who performed the task of fractioning the original sequences for the experiment described in section 3.2.Gallego Albiach, V.; Carneiro, PCF.; Mazzeo, I.; Vilchez Olivencia, MC.; Peñaranda, D.; Soler, C.; Pérez Igualada, LM.... (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology. 79(7):1034-1040. https://doi.org/10.1016/j.theriogenology.2013.01.019S1034104079

Exploring correlations between sex steroids and fatty acids and their potential roles in the induced maturation of the male European eel

[EN] The present study was undertaken to evaluate the correlations between the fatty acids in the liver and testis and the plasma levels of the hormonal steroids used during eel spermatogenesis, in order to clarify the physiological roles fatty acids play in the spermatogenetic process. The stages of testis development (S1-S5) were assessed by histological observations in order to classify the different phases of hormonally-induced spermatogenesis and evaluate the possible relationships between the hormones and fatty acids in each stage. The highest plasma levels of 17 beta-estradiol (E2), testosterone (T) and 11-ketotestosterone (11KT) were found in S1, when spermatogonial proliferation occurs. A correlation was found between 17 alpha-20 beta-dihydroxy-4-pregnen-3- one (DHP) levels and some fatty acids during the proliferation and growing phases (S1-2), suggesting that DHP might modulate lipid metabolism in the liver during early spermatogenesis. The DHP levels increased significantly during the growing phase (S2) and remained at high levels throughout the subsequent development stages (S3-S5). Similar to results found in mammals, our results show that in the eel there are regulatory mechanisms, including eicosapentaenoic acid (20:5-n3, EPA) and docosahexaenoic acid (22:6-n3, DHA), which act as modulators in the synthesis of androgens, particularly during the final phase of sperm maturation. Our results suggest that the fact that EPA, ARA and DHA concentrations in the eel testis remain constant/stable during spermiation could be related to the subsequent union of the spermatozoa and the egg. The findings from this research provide new insights for further studies about the possible effect of steroids on desaturase activity and highlight the importance of the effect of lipid metabolism during male eel spermatogenesis. (C) 2014 Elsevier B.V. All rights reserved.Funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL). D. S. P. had a contract co-financed by MICINN and UPV (PTA2011-4948-I) and received a Shortterm Scientific Mission grant from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) to carry out the steroids analyses in Norway.Baeza Ariño, R.; Peñaranda, D.; Vilchez Olivencia, MC.; Tveiten, H.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2015). Exploring correlations between sex steroids and fatty acids and their potential roles in the induced maturation of the male European eel. Aquaculture. 435:328-335. https://doi.org/10.1016/j.aquaculture.2014.10.016S32833543

Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species

[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.This study was funded by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the Spanish Personnel Research Training Program to carry out this research in the Misaki Marine Biological Station (Miura, Japan).Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2014). Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species. Theriogenology. 82(5):668-676. https://doi.org/10.1016/j.theriogenology.2014.05.026S66867682

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation

[EN] The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes. (C) 2012 Elsevier Inc. All rights reserved.This work was financed by the Spanish Ministry of Science and Innovation (MICINN; AGL2007-64040-C03-00, Project SELECTBREAM). V. Gallego and I. Mazzeo were supported by predoctoral scholarships financed by the Spanish MICINN and Generalitat Valenciana, respectively. D.S. Penaranda had a postdoctoral grant from UPV.Gallego Albiach, V.; Peñaranda, D.; Marco Jiménez, F.; Mazzeo, I.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2012). Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation. Theriogenology. 77(6):1078-1087. https://doi.org/10.1016/j.theriogenology.2011.10.010S1078108777

Using specific recombinant gonadotropins to induce spermatogenesis and spermiation in the European eel (Anguilla anguilla)

[EN] New specific European eel (Anguilla anguilla) recombinant gonadotropins (aarGths) produced in the ovarian cells of Chinese hamsters (CHO) were used to induce maturation in captive male eels. In the first experiment, five different hormonal treatments were assayed: one group was given a constant dose of recombinant European eel follicle-stimulating hormone (aarFsh; 4 ¿g/fish) for 9 weeks, and the second group received a constant dose of recombinant European eel luteinizing hormone (aarLh; 2 ¿g/fish) also for 9 weeks. The other three groups were injected with different combinations of both aarGths (some doses constant, some variable). All five treatments stimulated androgen synthesis, but the increase was more pronounced in the fish treated with a combination of both aarGths. Unlike aarLh, aarFsh alone was able to induce spermiation, the best results were achieved in the fish that were treated with a constant dose of aarFSH and an increasing dose of aarLH, with spermiation being induced (20% motile cells) despite the fact that these fish were immature at the start of the experiment. In order to improve sperm quality, a second experiment was performed. Immature males received three constant doses of aarFsh (2.8, 1.4 or 0.7 ¿g/fish) and increasing doses of aarLh (every 3 weeks; 1, 2, 6 ¿g/fish). All the treatments induced spermiation, however the best sperm quality (with ¿50% motile cells) was observed in the males treated with the highest dose of aarFsh. In conclusion, these specific recombinant gonadotropins have demonstrated their capacity to induce spermatogenesis and spermiation in vivo in a teleost fish, the European eel.This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (EN IMPRESS), the Spanish Ministry of Economy, Industry and Competitiveness (Project REPRO-TEMP; AGL2013-41646-R), Generalitat Valenciana (PROMETEOII/2014/051), VLC/CAMPUS Program (SP20140630) and the COST Office (COST Action FA1205: AQUAGAMETE). VG has a postdoc grant from the UPV (PAID-10-14). We would like to thank M.S. Ibanez for carrying out the ELISAs.Peñaranda, D.; Gallego Albiach, V.; Rozenfeld, C.; Herranz-Jusdado, JG.; Pérez Igualada, LM.; Gómez, A.; Giménez, I.... (2018). Using specific recombinant gonadotropins to induce spermatogenesis and spermiation in the European eel (Anguilla anguilla). Theriogenology. 107:6-20. https://doi.org/10.1016/j.theriogenology.2017.11.002S62010

Study of pufferfish (Takifugu niphobles) sperm: development of methods for short-term storage, effects of different activation media and role of intracellular changes in Ca2+ and K+ in the initiation of sperm motility

[EN] The first goal of this study was the development of a short-term storage method for pufferfish (Takifugu niphobles) sperm. In this respect, the best results were obtained by diluting the sperm in a seminal-like solution and keeping it in a Petri dish in chilled storage (4 degrees C). This method was successful in preserving sperm quality parameters without resulting in differences in fresh sperm for a relatively long-term period (7 days), for use in aquaculture matters. The addition of bovine serum albumin (BSA) to the medium did not improve the results. On the other hand, both the osmolality and the ion composition of the media are essential factors which can modulate the sperm motility parameters. The osmolality of the activating medium was the most important factor in triggering pufferfish sperm motility, and osmolalities of 750-825 mOsm/kg were necessary to initiate this process, demonstrating that it appears to be a dose-independent mechanism. Regarding the ion composition of the activation media, this study has shown that despite the spermatozoa being able to initiate movement without any ion in the activation medium, the absence of ions can negatively affect the kinetic parameters of the spermatozoa. Finally, in natural conditions (seawater), the activation of sperm motility generates intracellular increases in Ca2+ and K+, suggesting that these ions play an essential role in the activation mechanism of pufferfish sperm. (C) 2013 Elsevier B.V. All rights reserved.This study was funded by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) from the Spanish Personnel Research Training Programme to carry out this study in the Misaki Marine Biological Station (Miura, Japan).Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2013). Study of pufferfish (Takifugu niphobles) sperm: development of methods for short-term storage, effects of different activation media and role of intracellular changes in Ca2+ and K+ in the initiation of sperm motility. Aquaculture. 414:82-91. https://doi.org/10.1016/j.aquaculture.2013.07.046S829141

Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles)

[EN] The use of high quality gametes from both males and females during in vitro fertilization (IVF) trials is an essential step in order to achieve high fertilization and hatching rates. Although aquaculture hatcheries have focused more on egg rather than spermatozoa quality, some studies have demonstrated that sperm quantity and quality have a great influence both on fertilization/hatching success and the subsequent development of the embryo and larvae. In this study we have demonstrated that sperm/egg ratio and sperm quality are factors strongly related to each other in the pufferfish (Takifugu niphobles). Our results suggest that both factors should be taken into account as unique interrelated elements, making possible to obtain high fertilization rates using a successful combination of small amount of high quality sperm or high amount of low quality sperm. In addition, coefficients of correlation and determination among all the sperm motion parameters provided by a CASA system and fertilization/hatching rates were estimated for the first time in a marine species. Positive significant correlations were found in some parameters such as total and progressive motility (0.68 and 0.7 respectively). However, curvilinear velocity (VCL), straight line velocity (VSL) and average velocity (VAP) showed the highest coefficients of correlation (0.82, 0.8, and 0.81, respectively). In this respect, spermatozoa velocity appears to be a key factor in the fertilization process, especially when the number of spermatozoa per egg is limited in the aqueous environment. (C) 2013 Elsevier B.V. All rights reserved.Funded by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2010-16009). Victor Gallego has a predoctoral grant (MINECO; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the MINECO's Spanish Personnel Research Training Programme to carry out this research in the Misaki Marine Biological Station (Miura, Japan). We would like to thank to Dr. Kurokawa for the help and knowledge supplied during this study.Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2013). Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles). Aquaculture. 416:238-243. https://doi.org/10.1016/j.aquaculture.2013.08.035S23824341

Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results

[EN] Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility activation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca2+ and K+ sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility activation in marine fish, in which internal fluctuations of Ca2+ and K+ seem to participate in sperm activation. In addition, fluorescent images showed that both Ca2+ and K+ were concentrated in the apical area of the sperm head, which corresponds to the location of the eel sperm mitochondria, suggesting this organelle plays an important role in sperm motility activation. (C) 2013 Elsevier B.V. All rights reserved.Funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no 245257 (Pro-Eel) and the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the Spanish Personnel Research Training Programme to carry out this study in the Universidad de Leon (Leon, Spain). Ilaria Mazzeo had a predoctoral grant from GVA. David S. Penaranda has a contract co-financed by MICINN and UPV (PTA2011-4948-I). F. Martinez-Pastor was supported by the Ramon y Cajal program (RYC-2008-02560, MICINN).Gallego Albiach, V.; Martínez Pastor, F.; Mazzeo, I.; Peñaranda, D.; Herraez, P.; Asturiano Nemesio, JF.; Pérez Igualada, LM. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture. 418:155-158. https://doi.org/10.1016/j.aquaculture.2013.10.022S15515841