Efecto del regimen térmico en la calidad de los huevos y el desarrollo embrionario de la anguila europea (Anguilla anguilla)

[ES] Se ha evaluado el efecto de dos regímenes térmicos (constante y variable) aplicados durante la maduración sexual de hembras de anguila, en la calidad de las puestas y el desarrollo embrionario posterior. Se analizan parámetros preovulatorios, parámetros de calidad de los huevos (porcentaje de huevos flotantes, tasa de fecundación) y los estados de[EN] Two thermal regimes (constant and variable) were applied during the sexual maturation of female European eels, and their effects on preovulatory parameters, egg quality (percentage of floating eggs, fertilization rate) and embryo development were evaluated. The results are discussed taking in account the current bibliography.Vilchez Olivencia, MC. (2013). Efecto del regimen térmico en la calidad de los huevos y el desarrollo embrionario de la anguila europea (Anguilla anguilla). http://hdl.handle.net/10251/35849Archivo delegad

Role of calcium on the initiation of sperm motility in the European eel

[EN] Sperm from European eel males treated with hCG(rec), was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCI + EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+](i) fluorescence (and in [Na+](i) in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCI + EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+](i) levels did not increase at all. This result strongly suggests that increasing [Ca2+](i) is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+](i) concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm. (c) 2015 Elsevier Inc All rights reserved.Funded from the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vilchez has a predoctoral grant from UPV PAID Program (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia, GRISOLIA/2012/006), Victor Gallego has a postdoctoral contract from UPV (PAID-10-14), and David S. Penaranda was supported by MICINN (PTA2011-4948-I) and UPV (PTA2011-4948-I). Grants to attend meetings were received from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Pérez Igualada, LM.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Morini, M.; Peñaranda, D.; Asturiano Nemesio, JF. (2016). Role of calcium on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 191:98-106. https://doi.org/10.1016/j.cbpa.2015.10.009S9810619

Relationship between sperm quality parameters and fatty acid composition of the muscle, liver and testis of European eel

[EN] This study looks at the correlations that fatty acids have with different tissues in the European eel (Anguilla anguilla L.) during hormonally-induced sexual maturation, with different sperm quality parameters. In order to evaluate the different dynamics of the use of fatty acids, a categorization of the results from each sperm quality parameter (volume, concentration, motility and velocity) was performed. Low and moderate correlations were observed between muscle tissue and some sperm quality parameters but no high correlations were found. Eicosapentaenoic add (20:5n3, EPA) in the liver seems to have a role in determining the volume of sperm produced. This can be explained by the fact that EPA is a major requirement in the early phases of sperm production (probably as a component of the spermatozoal membrane). In addition, the levels of alpha-linolenic acid (18:3-n3, ALA) and linoleic acid (18:2-n6, LA) in the liver decreased when sperm motility increased. In all the tissues, a negative correlation was observed between arachidonic acid (20:4n-6, ARA) and the different sperm velocity parameters. The fact that an increase in the consumption of ARA coincides with an increase in the speed of spermatozoa, highlights the important role that this fatty acid plays not only in sperm production, but also in sperm velocity. All this information could prove useful in the development of suitable broodstock diets to improve sperm quality and subsequently, the larval development of this species. (C) 2014 Elsevier Inc All rights reserved.This work was funded by the European Community's Seventh Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no. 245257 (PRO-EEL), and COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez had predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). Rosa Baeza was supported by a contract funded by PRO-EEL. Authors want to thank Vicente Javier Moya Salvador for his technical assistance with gas chromatography analyses.Baeza Ariño, R.; Mazzeo, I.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Peñaranda, D.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2015). Relationship between sperm quality parameters and fatty acid composition of the muscle, liver and testis of European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 181:79-86. https://doi.org/10.1016/j.cbpa.2014.11.022S798618

Effect of thermal regime on fatty acid dynamics in male European eels (Anguilla anguilla) during hormonally-induced spermatogenesis

[EN] Little is known about the role of fat and fatty acids in European eel spermatogenesis. The aim of this research was to study the changes in fat content and to carry out a quantitative analysis of the fatty acid composition of the muscle, liver and gonad of European male eels during hormonally induced sexual maturation. Three different thermal regimes were used (two variable: T10 and T15; and one constant: T20) to replicate the changes in temperature that these fish experience during their transoceanic reproductive migration. Spermatogenesis was reached earlier in treatment T20, suggesting that spermatogenesis in the European eel is closely regulated by water temperature. Although eels lose body mass due to the period of fasting that accompanies gonadal growth, no significant changes were found in the fat and fatty acid content of the muscle during the experimental period. With regard to the liver, the levels of palmitic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids increased significantly at the start of the spermiation process in all the thermal treatments. In the testis, levels of EPA, arachidonic acid (ARA) and DHA remained constant during the maturation process whereas the levels of the rest of the fatty acids decreased significantly. The stability of the ARA and EPA levels in the testis may have a physiological significance, whereas the stability of the DHA levels may have a structural significance. The results suggest that the progression of spermiation is influenced by water temperature and demonstrate the importance of EPA, ARA and DHA in European eel reproduction. This study demonstrates that complementary research focusing on the lipid composition of commercial diets could improve the sperm quality of this species.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). Victor Gallego, Ilaria Mazzeo and M. Carmen Vilchez had predoctoral grants from the Spanish Ministry of Science and Innovation (MICINN), Generalitat Valenciana, and UPV PAID Programme (2011-S2-02-6521), respectively. David S. Penaranda was supported by a contract co-financed by MICINN and UPV (PTA2011-4948-I). Rosa Baeza was supported by contract financed by PRO-EEL. The authors want to thank Vicente Javier Moya Salvador for his technical assistance with gas chromatography analyses.Baeza Ariño, R.; Mazzeo, I.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Peñaranda, D.; Pérez Igualada, LM.; Asturiano Nemesio, JF. (2014). Effect of thermal regime on fatty acid dynamics in male European eels (Anguilla anguilla) during hormonally-induced spermatogenesis. Aquaculture. 430:86-97. https://doi.org/10.1016/j.aquaculture.2014.03.045S869743

Molecular markers of oocyte differentiation in European eel during hormonally induced oogenesis

[EN] Reproduction in captivity is a key study issue in Anguilla anguilla as a possible solution for its dwindling population. Understanding the mechanisms controlling the production of ribosomal building blocks during artificially induced oocyte maturation could be particularly interesting. Transcription levels of ribosomal biogenesis associated genes could be used as markers to monitor oogenesis. Eels from the Albufera Lagoon were injected with carp pituitary extract for 15 weeks and ovaries in previtellogenic (PV) stage (non-injected), in early-, mid-, late-vitellogenesis (EV, MV, LV), as well as in migratory nucleus stage (MN) were analysed. 5S rRNA and related genes were highly transcribed in ovaries with PV oocytes. As oocytes developed, transcriptional levels of genes related to 5S rRNA production (gtf3a), accumulation (gtf3a, 42sp43) and nucleocytoplasmic transport (tp15, tp111) and the 5S/18S rRNA index decreased (PV > EV > MV > LV > MN). On the contrary, 18S rRNA was at its highest at MN stage while ubtf1 in charge of activating RNA-polymerase I and synthesising 18S rRNA behaved as 5S related genes. Individuals that did not respond (NR) to the treatment showed 5S/18S index values similar to PV females, while studied genes showed EV/LV-like transcription levels. Therefore, NR females fail to express the largest rRNAs, which could thus be taken as markers of successful vitellogenesis progression. In conclusion, we have proved that the transcriptional dynamics of ribosomal genes provides useful tools to characterize induced ovarian development in European eels. In the future, such markers should be studied as putative indicators of response to hormonal treatments and of the quality of obtained eel oocytes.This work was supported by EU 7th Framework Programme (PRO EEL, grant agreement no. 245257 & AQUAGAMETE COST Action FA1205), Spanish MINECO (AGL2012-33477 and AGL2015-63936-R), Basque Government (S-PE12UN086 & IT810-13) & UPV/EHU (UFI 11/37). Some fish were supplied by the Hunting and Continental Fishing Service of Generalitat Valenciana. I.R.B holds a PhD fellowship of the Basque Government.Rojo-Bartolomé, I.; Martínez-Miguel, L.; Lafont, A.; Vilchez Olivencia, MC.; Asturiano Nemesio, JF.; Pérez Igualada, LM.; Cancio, I. (2017). Molecular markers of oocyte differentiation in European eel during hormonally induced oogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 211:17-25. https://doi.org/10.1016/j.cbpa.2017.05.018S172521

Nuclear and membrane progestin receptors in the European eel: characterization and expression in vivo through spermatogenesis

[EN] Characterization of all the progestin receptor genes (PRs) found in the European eel has been performed. There were five membrane PRs (mPRs): mPR alpha (alpha), mPRAL1 (alpha-likel), mPRAL2 (alpha-like2), mPRy (gamma), mPR delta (delta) and two nuclear PRs (nPRs or PGRs): pgr1 and pgr2. In silico studies showed that the C and E(F) domains of Pgr are well conserved among vertebrates whereas the A/B domain is not. Phylogeny and synteny analyses suggest that eel duplicated pgr (pgr1 and pgr2) originated from the teleost-specific third whole genome duplication (3R). mPR phylogeny placed three eel mPRs together with the mPRce Glade, being termed mPRet, mPRAL1 and mPRAL2, while the other two eel mPRs clustered with mPRy and mPRS clades, respectively. The in vivo study showed differential expression patterns along the brain-pituitary-gonad axis. An increase in nPR transcripts was observed in brain (in pgrl) and pituitary (in pgrl and pgr2) through the spermatogenesis, from the spermatogonia B/spermatocyte stage to the spermiation stage. In the testis, mPRy, mPRS and pgr2 transcripts showed the highest levels in testis with A spermatogonia as dominant germ cell, while the highest mPRce, mPRAL1 and mPRAL2 transcripts were observed in testis from spermiating males, where the dominant germ cell were spermatozoa. Further studies should elucidate the role of both nuclear and membrane progestin receptors on eel spermatogenesis.This work was supported by the Spanish Ministry of Science and Innovation (SPERMOT project; AGL2010-16009; REPRO-TEMP project, AGL2013-41646-R), and IMPRESS (Marie Sklodowska Curie Actions Innovative Training Network; Grant agreement no: 642893). M.C. Vilchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), M. Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia), D.S. Penaranda was supported by MICINN and UPV (PTA2011-4948-I). Grants to attend meetings were funded by COST Office (COST Action FA1205: AQUAGAMETE).Morini, M.; Peñaranda, D.; Vilchez Olivencia, MC.; Nourizadeh-Lillabadi, R.; Lafont, A.; Dufour, S.; Asturiano Nemesio, JF.... (2017). Nuclear and membrane progestin receptors in the European eel: characterization and expression in vivo through spermatogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 207:79-92. https://doi.org/10.1016/j.cbpa.2017.02.009S799220

Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCZ1) increase through induced spermatogenesis in European eel

[EN] Activation at fertilization of the vertebrate egg is triggered by Ca2+ waves. Recent studies suggest the phospholipase C zeta (PLC zeta), a sperm-specific protein, triggers egg activation by an 1P3-mediated Ca2+ release and allow Ca2+ waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLC zeta (PLC zeta 1). It is 1521bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLC zeta orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plc zeta was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plc zeta 1 expression in the pituitary gland. Testis plc zeta 1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLC zeta inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes. (C) 2015 Elsevier Inc. All rights reserved.Funded by the SPERMOT project (Spanish Ministry of Science and Innovation, MICINN; AGL2010-16009). M.C. Vílchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), Marina Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolía, GRISOLIA/2012/006), Victor Gallego has a postdoctoral grant (UPV; PAID-10-14), and David S. Peñaranda was a contract cofinanced by MICINN and UPV (PTA2011-4948-I). Grants to attend meetings from COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE).Morini, MAM.; Peñaranda, D.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Nourizadeh-Lillabadi, R.; Asturiano Nemesio, JF.; Weltzien, F.... (2015). Transcript levels of the soluble sperm factor protein phospholipase C zeta 1 (PLCZ1) increase through induced spermatogenesis in European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 187:168-176. https://doi.org/10.1016/j.cpba.2015.05.028S16817618

Temperature modulates testis steroidogenesis in European eel

[EN] This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10 degrees C (first 4 weeks), 15 degrees C (next 3 weeks) and 20 degrees C (last 6 weeks); T15: 15 degrees C (first 4 weeks) and 20 degrees C (last 9 weeks); and T20: constant 20 degrees C for the duration of the experiment. At 10 degrees C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was >= 15 degrees C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11 beta HSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2).Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was >= 15 degrees C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration. (C) 2016 Elsevier Inc. All rights reserved.This study was funded by the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no245257 (PRO-EEL), VLC/CAMPUS Program (SP.20140630) and by the MINECO (REPRO-TEMP; AGL2013-41646-R). V.G. and I.M. had predoctoral grants from MINECO (BES-2009-020310) and Generalitat Valenciana, respectively. M.C.V. and M.M. have predoctoral grants from UPV (2011-S2-02-6521) and Generalitat Valenciana (Programa Grisolia), respectively. D.S.P. was supported by MICINN and UPV (PTA2011-4948-I) and was granted with a Short-Term Scientific Mission to make the steroids analyses in Tromso by COST Office (COST Action FA1205: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry; AQUAGAMETE).Peñaranda, D.; Morini, M.; Tveiten, H.; Vilchez Olivencia, MC.; Gallego Albiach, V.; Dirks, R.; Van Den Thillart, GE.... (2016). Temperature modulates testis steroidogenesis in European eel. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 197:58-67. https://doi.org/10.1016/j.cbpa.2016.03.012S586719

Identification of the major proteins present in the seminal plasma of European eel, and how hormonal treatment affects their evolution. Correlation with sperm quality

[EN] By first time, 2DE protein profile of European eel seminal plasma has been determined. 14 different proteins corresponding to 9 major families were identified in seminal plasma, through hormonal treatment. Some of them play a part in sperm maturation, including carbonic anhydrase which is responsible for modulating the pH of seminal plasma, and warm temperature acclimation protein, which may play an important role in the final maturation of this species, due to the warm temperature of their spawning ground (in the Sargasso Sea). Sperm samples were classified into three motility categories depending on the percentage of motile cells, I: 0-25%, II: 25-50% and 111: >50%. Different protein profiles were observed depending on the sperm motility categories, specifically, with the apolipoproteins and complement C3. Higher numbers of proteins from the apolipoprotein family were registered at lower motilities; whereas the complement C3-like family was higher in the samples with the highest percentage of motile cells. These results suggest that the proteins linked to the transportation of lipids (apolipoprotein) and to the immune system (complement C3) may carry out their functions at different stages of spermatogenesis. Using SDS-PAGE analysis, 13 bands were identified, most of which migrated between 20 to 60 kDa. In the last weeks of treatment significant increases were observed in the percentage of motile spermatozoa, curvilinear velocity and beat cross frequency. This improvement in sperm quality coincided with a higher amount of proteins located at 19 KDa, therefore, this protein could be involved in sperm motility of the European eel. (C) 2016 Published by Elsevier Inc.Funded from the REPRO-TEMP project (Spanish Ministry of Science and Innovation, MICINN; AGL2013-41646-R). and COST Office (COST Action FA1205: AQUAGAMETE). M.C.V. and V.G. have pre- and post-doctoral grants from UPV PAID Programme (2011-S2-02-6521 and 10-14 respectively). Valenciana de Acuicultura, S.A. (Puzol, Spain) supplied the eels used in this study.Vilchez Olivencia, MC.; D. Pla; Gallego Albiach, V.; Sanz, L.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Calvete, JJ.... (2016). Identification of the major proteins present in the seminal plasma of European eel, and how hormonal treatment affects their evolution. Correlation with sperm quality. Comparative Biochemistry and Physiology - Part A: Molecular and Integrative Physiology. 201:37-45. https://doi.org/10.1016/j.cbpa.2016.06.025S374520

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla)

[EN] Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14 +/- 1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol + 15% propylene-glycol), cooling device: Cryotop, 2 mu l droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2 mu l of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54 +/- 1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.The work was funded by the NKFI (previously OTKA) project number K-109847 and by a Short-term Scientific Mission awarded to E. Kasa by the COST Office (Food and Agriculture COST Action FA1205: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry; AQUAGAMETE). The work was supported by the project Research Center of Excellence - 9878-3/2016/FEKUT of the Ministry of Human Resources of Hungary and the project EUREKA_HU_12-1-2012-0056 (PERCAHATCH).Kása, E.; Bernáth, G.; Kollár, T.; Zarski, D.; Lujic, J.; Marinovic, Z.; Bokor, Z.... (2017). Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla). General and Comparative Endocrinology. 245:102-107. https://doi.org/10.1016/j.ygcen.2016.05.010S10210724