First production of larvae using cryopreserved sperm. Effects of preservation temperature and cryopreservation on European eel sperm fertilization capacity

[EN] Contents Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20 degrees C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20 degrees C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae (cryolarvae') were obtained 55h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.This study was part of the project: 'Reproduction of European Eel: Towards a Self-sustained Aquaculture' (PRO-EEL) European Community's 7th Framework Programme under the Theme 2 'Food, Agriculture and Fisheries, and Biotechnology', Grant Agreement no245257. Juan F. Asturiano and Luz Perez had a grant to staying in Denmark from Programa de Apoyo a la Investigacion y Desarrollo (PAID-00-11) of the Universitat Politecnica de Valencia. We thank Christian Graver and Lars B. Tybjerg for assistance duringAsturiano Nemesio, JF.; Sorensen, S.; Pérez Igualada, LM.; Lauesen, P.; Tomkiewicz, J. (2016). First production of larvae using cryopreserved sperm. Effects of preservation temperature and cryopreservation on European eel sperm fertilization capacity. Reproduction in Domestic Animals. 51(4):485-491. doi:10.1111/rda.12706S48549151

Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review

Protocols for the cryopreservation of fish gametes have been developed for many different fish species, in special, freshwater salmonids and cyprinids. Methods for sperm freezing have progressed during the last decades due to the increasing number of potential applications: aquaculture (genetic improvement programs, broodstock management, helping with species having reproductive problems), biotechnology studies using model fish species (preservation of transgenic or mutant lines), cryobanking of genetic resources from endangered species, etc. This mini-review tries to give an overview of the present situation of this area of research, identifying the main challenges and perspectives, redirecting the reader to more in-depth reviews and papers. (C) 2016 Elsevier Inc. All rights reserved.European Training Network IMPRESS (Marie Sklodowska-Curie Actions) from PROMAR programme [642893]COST Office (Food and Agriculture COST Action FA1205: AQUAGAMETE) from PROMAR programmeResearch Centre of Excellence from PROMAR programme [9878-3/2016/FEKUT, KMR_12-1-2012-0436]NKFIH (OTKA) from PROMAR programme [109847]KLING from PROMAR programme [31-03-05FEP-73]CRIOBIV from PROMAR programme [31-03-05-FEP-59]REPLING from PROMAR programme [31-03-05-FEP-69]info:eu-repo/semantics/publishedVersio

Japanese eel (Anguilla japonica Temminck & Schlegel, 1846) propagation by using cryopreserved sperm samples

[EN] The aim of this study was to test the artificial seminal plasma (ASP) as the extender as well as methanol as the cryoprotectant for cryopreservation, and to collect information on the fertilizing capacity of cryopreserved sperm samples. Eggs from Japanese eel, Anguilla japonica, females were control-fertilized with native sperm diluted with ASP and cryopreserved sperm (ASP as extender [in 1:100 ratio] and 10% methanol in v/v final concentration). No statistical differences (p<.05) were among the measured parameters (hatching, survival after 10days post-hatch, malformation rates) between the two groups, thus this cryopreservation method can be used effectively for artificial propagation of the Japanese eel, Anguilla japonica.Tokyo University of Agriculture Strategic Research Program (TUA-SRP); Mohamed bin Zayed Species Conservation Fund, Grant/ Award Number: 162512761; Research Centre of Excellence, Grant/Award Number: 11476-3/ 2016/FEKUTMüller, T.; Matsubara, H.; Kubara, Y.; Horváth, Á.; Asturiano Nemesio, JF.; Urbányi, B. (2017). Japanese eel (Anguilla japonica Temminck & Schlegel, 1846) propagation by using cryopreserved sperm samples. Journal of Applied Ichthyology (Online). 33:550-552. https://doi.org/10.1111/jai.13316S55055233Abe, T., Ijiri, S., Adachi, S., & Yamauchi, K. (2010). Development of an in vitro culture system for producing eel larvae from immature ovarian follicles in Japanese eel Anguilla japonica. Fisheries Science, 76(2), 257-265. doi:10.1007/s12562-010-0216-8Asturiano, J., Sørensen, S., Pérez, L., Lauesen, P., & Tomkiewicz, J. (2016). First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity. Reproduction in Domestic Animals, 51(4), 485-491. doi:10.1111/rda.12706Kurokawa, T., Okamoto, T., Gen, K., Uji, S., Murashita, K., Unuma, T., … Tanaka, H. (2008). Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel,Anguilla japonica, at Completion of Yolk Resorption. Journal of the World Aquaculture Society, 39(6), 726-735. doi:10.1111/j.1749-7345.2008.00208.xMüller, T., Horváth, Á., Takahashi, E., Kolics, B., Bakos, K., Decsi, K., … Yamaha, E. (2012). Artificial hybridization of Japanese and European eel (Anguilla japonica×A. anguilla) by using cryopreserved sperm from freshwater reared males. Aquaculture, 350-353, 130-133. doi:10.1016/j.aquaculture.2012.04.007Ohta, H., Kagawa, H., Tanaka, H., Okuzawa, K., Iinuma, N., & Hirose, K. (1997). Fish Physiology and Biochemistry, 17(1/6), 163-169. doi:10.1023/a:1007720600588Okamoto, T., Kurokawa, T., Gen, K., Murashita, K., Nomura, K., Kim, S.-K., … Tanaka, H. (2009). Influence of salinity on morphological deformities in cultured larvae of Japanese eel, Anguilla japonica, at completion of yolk resorption. Aquaculture, 293(1-2), 113-118. doi:10.1016/j.aquaculture.2009.04.005Tanaka, S., Zhang, H., Horie, N., Yamada, Y., Okamura, A., Utoh, T., … Kurokura, H. (2002). Long-term cryopreservation of sperm of Japanese eel. Journal of Fish Biology, 60(1), 139-146. doi:10.1111/j.1095-8649.2002.tb02393.xUnuma, T., Kondo, S., Tanaka, H., Kagawa, H., Nomura, K., & Ohta, H. (2004). Determination of the rates of fertilization, hatching and larval survival in the Japanese eel, Anguilla japonica, using tissue culture microplates. Aquaculture, 241(1-4), 345-356. doi:10.1016/j.aquaculture.2004.08.00

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

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

Reproductive anatomy of Chondrichthyans: notes on specimen handling and sperm extraction. II. Sharks and chimaeras

[EN] Sperm extraction and artificial insemination may serve ex situ conservation initiatives for threatened sharks and related species. A comparison of the reproductive anatomy of eight chondrichthyans is presented in this study, emphasizing the important differences when performing these reproductive techniques. Additionally, we show how to obtain sperm samples from both living and dead specimens using cannulation, abdominal massage, or oviducal gland extraction. These tools can improve the success of breeding programs developed in aquaria and research facilities. The chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates on the planet. Given this situation, an additional strategy for the protection of these species could be the ex situ conservation projects developed in public aquaria and research centers. Nevertheless, to increase sustainability and to develop properly in situ reintroduction strategies, captive breeding techniques, such as sperm extraction and artificial insemination, should be developed. These techniques are commonly used in other threatened species and could be also used in chondrichthyans. However, the different reproductive morphologies found in this group can complicate both processes. Therefore, a comparison of the reproductive anatomy of eight distinct chondrichthyans, with an emphasis on those important differences when performing sperm extraction or artificial insemination, is carried out herein. Sharks and chimaeras belonging to the Scyliorhinidae, Carcharhinidae, Centrophoridae, Etmopteridae, Hexanchidae, and Chimaeridae families were obtained from commercial fisheries, public aquaria, and stranding events. In addition, the process of obtaining viable sperm samples through cannulation, abdominal massage, and oviducal gland extraction is described in detail for both living and dead animals.This research was partially funded by the Fundacion Biodiversidad (PRCV00683). P.G.-S. has a PhD contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ACIF 2018 (ACIF/2018/147). V.G. has a postdoc contract from the MICIU, Programa Juan de la Cierva-Incorporacion (IJCI-2017-34200).García-Salinas, P.; Gallego Albiach, V.; Asturiano, JF. (2021). Reproductive anatomy of Chondrichthyans: notes on specimen handling and sperm extraction. II. Sharks and chimaeras. Animals. 11(8):1-20. https://doi.org/10.3390/ani11082191S12011

Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. I. Rays and Skates

[EN] Many species of rays and skates are endangered, and ex situ conservation programs developed by research centers and public aquaria could improve this situation. To reproduce these species in captivity, scientists need to know how to extract their sperm and how to conduct their artificial insemination; however, the anatomical diversity of the reproductive organs of this group of animals is a handicap. A comparison of the reproductive anatomies of 11 distinct species is presented here, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. In addition, the process of obtaining sperm samples from both live and dead males is described in detail, using both cannulation and abdominal massage. The superorder Batoidea (rays, skates, and relatives), constitutes one of the most threatened group of vertebrates. Strengthening ex situ conservation programs developed in research centers and public aquaria could be a way of addressing this situation. However, captive breeding programs must be improved to prevent the capture of wild animals and to develop proper in situ reintroduction strategies. Sperm extraction and artificial insemination are two techniques commonly used in other threatened species, which could also be used in rays and the like. However, the different reproductive morphologies present within this group of animals may hamper both processes. Here, we present a comparison of the reproductive anatomies of 11 distinct batoid species, emphasizing the important differences between the species when performing sperm extraction or artificial insemination. Both male and female animals, belonging to the Rajidae, Dasyatidae, Torpedinidae and Myliobatidae families, from the Mediterranean Sea were studied. In addition, we describe the procedure to extract sperm using both cannulation and abdominal massage, either from live or dead batoids Finally, the obtention of motile sperm recovered from the oviducal gland of females is described. These techniques generate a new range of possibilities for the conservation of these threatened species.This research was partially funded by the Fundacion Biodiversidad (PRCV00683). PGS has a PhD contract from the European Union through the Operational Program of the European Social Fund (ESF) of the Comunitat Valenciana 2014-2020 ACIF 2018 (ACIF/2018/147). VG has a postdoc contract from the MICIU, Programa Juan de la Cierva-Incorporacion (IJCI-2017-34200).García-Salinas, P.; Gallego Albiach, V.; Asturiano, JF. (2021). Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. I. Rays and Skates. Animals. 11(7):1-16. https://doi.org/10.3390/ani11071888S11611

Intracellular pH regulation and sperm motility in the European eel

[EN] Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH(i)) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pH(i) range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH(e)), as well as between the pH(i) and the pH of the diluent. The change post-activation in seawater (ASW) depended on the initial pH(e) of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH(e) 8, and no pH(i) variation when pH(e) was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH(i) changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. (C) 2020 Elsevier Inc. All rights reserved.Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS). Victor Gallego has a postdoc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-2017-34200Pérez Igualada, LM.; Gallego Albiach, V.; Asturiano, JF. (2020). Intracellular pH regulation and sperm motility in the European eel. Theriogenology. 145:48-58. https://doi.org/10.1016/j.theriogenology.2020.01.026S4858145Nishigaki, T., José, O., González-Cota, A. L., Romero, F., Treviño, C. L., & Darszon, A. (2014). Intracellular pH in sperm physiology. Biochemical and Biophysical Research Communications, 450(3), 1149-1158. doi:10.1016/j.bbrc.2014.05.100Ohta, H., Ikeda, K., & Izawa, T. (1997). Increases in concentrations of potassium and bicarbonate ions promote acquisition of motility in vitro by Japanese eel spermatozoa. The Journal of Experimental Zoology, 277(2), 171-180. doi:10.1002/(sici)1097-010x(19970201)277:23.0.co;2-mOda, S., & Morisawa, M. (1993). Rises of intracellular Ca2+ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motility and the Cytoskeleton, 25(2), 171-178. doi:10.1002/cm.970250206TANAKA, S., UTOH, T., YAMADA, Y., HORIE, N., OKAMURA, A., AKAZAWA, A., … KUROKURA, H. (2004). Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel. Fisheries Science, 70(5), 780-787. doi:10.1111/j.1444-2906.2004.00871.xGallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D. S., Herráez, M. P., Asturiano, J. F., & Pérez, L. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture, 418-419, 155-158. doi:10.1016/j.aquaculture.2013.10.022Vílchez, M. C., Morini, M., Peñaranda, D. S., Gallego, V., Asturiano, J. F., & Pérez, L. (2017). Role of potassium and pH on the initiation of sperm motility in the European eel. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 203, 210-219. doi:10.1016/j.cbpa.2016.09.024M�ri�n, T., Krasznai, Z., Balkay, L., Emri, M., & Tr�n, L. (1997). Role of extracellular and intracellular pH in carp sperm motility and modifications by hyperosmosis of regulation of the Na+/H+ exchanger. Cytometry, 27(4), 374-382. doi:10.1002/(sici)1097-0320(19970401)27:43.0.co;2-cPerez, L., Aturiano, J. F., Tomas, A., Zegrari, S., Barrera, R., Espinos, F. J., … Jover, M. (2000). Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. Journal of Fish Biology, 57(6), 1488-1504. doi:10.1111/j.1095-8649.2000.tb02227.xGallego, V., Carneiro, P. C. F., Mazzeo, I., Vílchez, M. C., Peñaranda, D. S., Soler, C., … Asturiano, J. F. (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology, 79(7), 1034-1040. doi:10.1016/j.theriogenology.2013.01.019Mylonas, C. C., Duncan, N. J., & Asturiano, J. F. (2017). Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture, 472, 21-44. doi:10.1016/j.aquaculture.2016.04.021Asturiano, J. F., Pérez, L., Garzón, D. L., Marco-Jiménez, F., Peñaranda, D. S., Vicente, J. S., & Jover, M. (2004). Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm. Fish Physiology and Biochemistry, 30(3-4), 283-293. doi:10.1007/s10695-005-1553-xThomas, J. A., Buchsbaum, R. N., Zimniak, A., & Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry, 18(11), 2210-2218. doi:10.1021/bi00578a012Krasznai, Z. (2003). Role of ion channels and membrane potential in the initiation of carp sperm motility. Aquatic Living Resources, 16(5), 445-449. doi:10.1016/s0990-7440(03)00054-8Miura, T., & Miura, C. I. (2003). Molecular control mechanisms of fish spermatogenesis. Fish Physiology and Biochemistry, 28(1-4), 181-186. doi:10.1023/b:fish.0000030522.71779.47Fechner, S., Alvarez, L., Bönigk, W., Müller, A., Berger, T. K., Pascal, R., … Kaupp, U. B. (2015). A K+-selective CNG channel orchestrates Ca2+ signalling in zebrafish sperm. eLife, 4. doi:10.7554/elife.07624Peñaranda, D., Pérez, L., Gallego, V., Barrera, R., Jover, M., & Asturiano, J. (2008). European Eel Sperm Diluent for Short-term Storage. Reproduction in Domestic Animals, 45(3), 407-415. doi:10.1111/j.1439-0531.2008.01206.xChauvaud, L., Cosson, J., Suquet, M., & Billard, R. (1995). Sperm motility in turbot, Scophthalmus marimus: initiation of movement and changes with time of swimming characteristics. Environmental Biology of Fishes, 43(4), 341-349. doi:10.1007/bf00001167Billard, R., Cosson, J., & Crim, L. W. (1993). Motility of fresh and aged halibut sperm. Aquatic Living Resources, 6(1), 67-75. doi:10.1051/alr:1993008Effer, B., Figueroa, E., Augsburger, A., & Valdebenito, I. (2013). Sperm biology of Merluccius australis: Sperm structure, semen characteristics and effects of pH, temperature and osmolality on sperm motility. Aquaculture, 408-409, 147-151. doi:10.1016/j.aquaculture.2013.05.040Inaba, K., Dréanno, C., & Cosson, J. (2003). Control of flatfish sperm motility by CO2and carbonic anhydrase. Cell Motility and the Cytoskeleton, 55(3), 174-187. doi:10.1002/cm.10119Lee, H. cheung, Johnson, C., & Epel, D. (1983). Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm. Developmental Biology, 95(1), 31-45. doi:10.1016/0012-1606(83)90004-0Babcock, D. F., Rufo, G. A., & Lardy, H. A. (1983). Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proceedings of the National Academy of Sciences, 80(5), 1327-1331. doi:10.1073/pnas.80.5.132

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 subpopulation pattern of eel sperm is affected by post-activation time, hormonal treatment and thermal regime

[EN] There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90 s), the thermal regimen (i.e. 10 degrees C (T10) or 15 degrees C (T15) and up to 20 degrees C, or constant at 20 degrees C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30 s is recommended as the standard time for motility acquisition. When eels were kept at 20 degrees C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8 +/- 1.3%; mean +/- s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0 +/- 1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.This study was funded by the European Community's 7th Framework Program under the Theme 2 'Food, Agriculture and Fisheries, and Bio-technology', grant agreement no. 245257 (PRO-EEL) and Generalitat Valenciana (ACOMP/2012/086). VG and MCV have predoctoral grants from the Spanish Ministry of Economy and Competitiveness (AGL2010-16009) and Universitat Politecnica de Valencia (UPV) PAID Program (2011-S2-02-6521), respectively. DSP was supported by a contract cofinanced by Ministry of Science and Innovation (MICINN) and UPV (PTA2011-4948-I). FM-P was supported by the Ramon y Cajal program (MICINN, RYC-2008-02560).Gallego Albiach, V.; Vilchez Olivencia, MC.; Peñaranda, D.; Pérez Igualada, LM.; Herraez, MP.; Asturiano Nemesio, JF.; Martinez-Pastor, F. (2015). The subpopulation pattern of eel sperm is affected by post-activation time, hormonal treatment and thermal regime. Reproduction, Fertility and Development. 27(3):529-543. https://doi.org/10.1071/RD13198S52954327