5th International Workshop on the Biology of Fish Gametes Evidences of a series growing in numbers and quality

Asturiano Nemesio, JF.; Horváth, Á.; Carnevali, O. (2017). 5th International Workshop on the Biology of Fish Gametes Evidences of a series growing in numbers and quality. General and Comparative Endocrinology. 245:1-4. doi:10.1016/j.ygcen.2017.03.004S1424

Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality

[EN] This article reviews the use of hormonal treatments to enhance sperm production in aquaculture fish and the methods available for evaluating sperm quality. The different types of testis development are examined and a brief review is presented of the endocrine regulation of spermatogenesis in fishes, including the increasing evidence of the existence of spermatozoa subpopulations. Hormonal manipulations are employed to induce spermatogenesis in species such as the freshwater eels, to synchronize maximal sperm volume to ovulation for in vitro fertilization and to enhance sperm production in species with poor spermiation. The hormones that are employed include gonadotropins (GtHs) of piscine or mammalian origin, and gonadotropin-releasing hormone agonists (GnRHa) administered by injections or controlled-release delivery systems, with or without dopaminergic inhibitors. Pheromones in the culture water and hormones added to the sperm in vitro have also been employed to enhance spermiation and sperm quality, respectively, in some fishes. Hormonal therapies usually do not affect sperm quality parameters, except in cases where fish fail to spermiate naturally or produce very small volumes of high-density sperm. Different parameters have been used to evaluate fish sperm quality, including sperm volume and density, spermatozoa motility and morphometry, and seminal plasma composition. The development of Computer-Assisted Sperm Analysis (CASA) systems made possible the estimation of a higher number of sperm motion parameters using an objective, sensitive and accurate technique. The development of Assisted Sperm Morphology Analysis (ASMA) software has introduced a new approach for sperm evaluation studies, demonstrating changes in the spermatozoa related to reproductive season, hormonal treatments or the cryopreservation processes, and how these may be related to changes in sperm motility and fertilization capacity. The article concludes with a few practical protocols for the enhancement of sperm production in aquaculture species.We would like to thank the COST Action FA1205 (AQUAGAMETE) titled “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” for initiating the production of this special issue and funding partially this article. JFA has been partly funded by the Spanish Ministry of Economy and Competitiveness (REPRO-TEMP; AGL2013-41646-R) and the European Training Network IMPRESS (Marie SklodowskaCurie Actions; Grant agreement n°: 642893). ND has been funded partly by the INIA-FEDER project RTA2011-00050, and CCM and ND have been supported also by the EU project DIVERSIFY (7FP-KBBE-2013-GA 602131).Mylonas, CC.; Duncan, NJ.; Asturiano Nemesio, JF. (2017). Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture. 472:21-44. https://doi.org/10.1016/j.aquaculture.2016.04.021S214447

The Fourth International Workshop on the Biology of Fish Gametes, 17-20 September 2013, Albufeira, Algarve, Portugal: Summary and conclusions

Rosenthal, H.; Asturiano Nemesio, JF.; Horváth, Á.; Martinez-Paramo, S. (2015). The Fourth International Workshop on the Biology of Fish Gametes, 17-20 September 2013, Albufeira, Algarve, Portugal: Summary and conclusions. Journal of Applied Ichthyology. 31(1):1-2. doi:10.1111/jai.12716S1231

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

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

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

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