Free Standard AU & NZ Shipping For All Book Orders Over $80!
Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen

V. Gallego A , M. C. Vílchez A , D. S. Peñaranda A , L. Pérez A , M. P. Herráez B , J. F. Asturiano A and F. Martínez-Pastor B C
+ Author Affiliations
- Author Affiliations

A Grupo de Acuicultura y Biodiversidad, Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València, Camino de Vera s/n. 46022, Valencia, Spain.

B Institute for Cattle Development and Animal Health Institute for Cattle Development and Animal Health (INDEGSAL) and Molecular Biology, University of León, 24071 León, Spain.

C Corresponding author. Email: felipe.martinez@unileon.es

Reproduction, Fertility and Development 27(3) 529-543 https://doi.org/10.1071/RD13198
Submitted: 23 June 2013  Accepted: 8 January 2014   Published: 4 March 2014

Abstract

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°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°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°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.

Additional keywords: computer-aided sperm analysis, European eel, motility activation.


References

Aarestrup, K., Okland, F., Hansen, M. M., Righton, D., Gargan, P., Castonguay, M., Bernatchez, L., Howey, P., Sparholt, H., Pedersen, M. I., and McKinley, R. S. (2009). Oceanic spawning migration of the European eel (Anguilla anguilla). Science 325, 1660.
Oceanic spawning migration of the European eel (Anguilla anguilla).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFGqt77O&md5=b16b8ef1ef076f29da3165fcdfba2812CAS | 19779192PubMed |

Al-Inany, H., Aboulghar, M. A., Mansour, R. T., and Proctor, M. (2005). Recombinant versus urinary gonadotrophins for triggering ovulation in assisted conception. Hum. Reprod. 20, 2061–2073.
Recombinant versus urinary gonadotrophins for triggering ovulation in assisted conception.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXmt1Gnu70%3D&md5=20a5064bc1de8dc73376d8dbd204a497CAS | 16024539PubMed |

Alavi, S. M. H., and Cosson, J. (2005). Sperm motility in fishes. I. Effects of temperature and pH: a review. Cell Biol. Int. 29, 101–110.
Sperm motility in fishes. I. Effects of temperature and pH: a review.Crossref | GoogleScholarGoogle Scholar |

Alavi, S. M. H., and Cosson, J. (2006). Sperm motility in fishes. (II) Effects of ions and osmolality: a review. Cell Biol. Int. 30, 1–14.
Sperm motility in fishes. (II) Effects of ions and osmolality: a review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhsF2ntr8%3D&md5=7c955f8c6de3eb47cb51750f9ff7b5c7CAS |

Asturiano, J. F., Sorbera, L. A., Ramos, J., Kime, D. E., Carrilo, M., and Zanuy, S. (2000). Hormonal regulation of the European sea bass reproductive cycle: an individualized female approach. J. Fish Biol. 56, 1155–1172.
Hormonal regulation of the European sea bass reproductive cycle: an individualized female approach.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXktlals74%3D&md5=b0bdef4a2825851b4afb273a04e29643CAS |

Asturiano, J. F., Sorbera, L. A., Ramos, J., Kime, D. E., Carrillo, M., and Zanuy, S. (2002). Group-synchronous ovarian development, spawning and spermiation in the European sea bass (Dicentrarchus labrax L.) could be regulated by shifts in gonadal steroidogenesis. Sci. Mar. 66, 273–282.

Asturiano, J. F., Pérez, L., Garzón, D. L., Marco-Jiménez, F., Peñaranda, D. S., Vicente, J. S., and Jover, M. (2004). Physio-chemical characteristics of seminal plasma and development of media and methods for the cryopreservation of European eel sperm. Fish Physiol. Biochem. 30, 283–293.
Physio-chemical characteristics of seminal plasma and development of media and methods for the cryopreservation of European eel sperm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXht1OqtrrO&md5=bcdc96317494b53ad8b7e8283775894aCAS |

Asturiano, J. F., Pérez, L., Garzón, D. L., Peñaranda, D. S., Marco-Jiménez, F., Martínez-Llorens, S., Tomás, A., and Jover, M. (2005). Effect of different methods for the induction of spermiation on semen quality in European eel. Aquacult. Res. 36, 1480–1487.
Effect of different methods for the induction of spermiation on semen quality in European eel.Crossref | GoogleScholarGoogle Scholar |

Asturiano, J. F., Marco-Jiménez, F., Pérez, L., Balasch, S., Garzón, D. L., Peñaranda, D. S., Vicente, J. S., Viudes-de Castro, M. P., and Jover, M. (2006). Effects of hCG as spermiation inducer on European eel semen quality. Theriogenology 66, 1012–1020.
Effects of hCG as spermiation inducer on European eel semen quality.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xot1Wrt7c%3D&md5=cb6fa2779de76fee3c9d5f0c021b3569CAS | 16595151PubMed |

Beirão, J., Soares, F., Herráez, M. P., Dinis, M. T., and Cabrita, E. (2009). Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level. Theriogenology 72, 1251–1261.
Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.Crossref | GoogleScholarGoogle Scholar | 19781754PubMed |

Beirão, J., Cabrita, E., Pérez-Cerezales, S., Martínez-Páramo, S., and Herráez, M. P. (2011). Effect of cryopreservation on fish sperm subpopulations. Cryobiology 62, 22–31.
Effect of cryopreservation on fish sperm subpopulations.Crossref | GoogleScholarGoogle Scholar | 21112321PubMed |

Breton, B., and Billard, R. (1977). Effects of photoperiod and temperature on plasma gonadotropin and spermatogenesis in the rainbow trout, Salmo gairdnerii Richardson. Ann. Biol. Anim. Biochim. Biophys. 17, 331–340.
Effects of photoperiod and temperature on plasma gonadotropin and spermatogenesis in the rainbow trout, Salmo gairdnerii Richardson.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE2sXks1Gmu74%3D&md5=e7c6cd9c57ba2eeee5259e5338ad3f6eCAS |

Crochet, J. R., Shah, A. A., Schomberg, D. W., and Price, T. M. (2012). Hyperglycosylated human chorionic gonadotropin does not increase progesterone production by luteinized granulosa cells. J. Clin. Endocrinol. Metab. 97, E1741–E1744.
| 1:CAS:528:DC%2BC38XhtlylsrvM&md5=3a50567828b88a89708073bdbc15b3f0CAS | 22745244PubMed |

de Paz, P., Alvarez-Rodriguez, M., Nicolas, M., Alvarez, M., Chamorro, C., Borragán, S., Martinez-Pastor, F., and Anel, L. (2012). Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos). Reprod. Domest. Anim. 47, 105–112.
Optimization of glycerol concentration and freezing rate in the cryopreservation of ejaculate from brown bear (Ursus arctos).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XislWmt70%3D&md5=7d06bda3569395cd3c11f8063dd2796bCAS | 21615800PubMed |

Domínguez-Rebolledo, A. E., Fernández-Santos, M. R., García-Alvarez, O., Maroto-Morales, A., Garde, J. J., and Martínez-Pastor, F. (2009). Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa. Theriogenology 72, 1073–1084.
Washing increases the susceptibility to exogenous oxidative stress in red deer spermatozoa.Crossref | GoogleScholarGoogle Scholar | 19733389PubMed |

Domínguez-Rebolledo, A. E., Martínez-Pastor, F., Bisbal, A. F., Ros-Santaella, J. L., García-Álvarez, O., Maroto-Morales, A., Soler, A. J., Garde, J. J., and Fernández-Santos, M. R. (2011). Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability. Reprod. Domest. Anim. 46, 393–403.
Response of thawed epididymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability.Crossref | GoogleScholarGoogle Scholar | 20678089PubMed |

Dzyuba, B. B., Van Look, K. J. W., Kholodnyy, V. S., Satake, N., Cheung, S., and Holt, W. V. (2008). Variable sperm size and motility activation in the pipefish, syngnathus abaster; adaptations to paternal care or environmental plasticity? Reprod. Fertil. Dev. 20, 474–482.
Variable sperm size and motility activation in the pipefish, syngnathus abaster; adaptations to paternal care or environmental plasticity?Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1czgtVOrtw%3D%3D&md5=6c8358b135459d9115aecaae55f6825fCAS | 18462609PubMed |

Fernández-Santos, M. R., Martínez-Pastor, F., García-Macías, V., Esteso, M. C., Soler, A. J., de Paz, P., Anel, L., and Garde, J. J. (2007). Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa. Theriogenology 67, 738–753.
Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa.Crossref | GoogleScholarGoogle Scholar | 17140651PubMed |

Feunteun, E. (2002). Management and restoration of European eel population (Anguilla anguilla): an impossible bargain. Ecol. Eng. 18, 575–591.
Management and restoration of European eel population (Anguilla anguilla): an impossible bargain.Crossref | GoogleScholarGoogle Scholar |

Gallego, V., Mazzeo, I., Vílchez, M., Peñaranda, D., Carneiro, P., Pérez, L., and Asturiano, J. (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 354–355, 7–16.
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.Crossref | GoogleScholarGoogle Scholar |

Gallego, V., Carneiro, P. C. F., Mazzeo, I., Vílchez, M. C., Peñaranda, D. S., Soler, C., Pérez, L., and Asturiano, J. F. (2013a). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology 79, 1034–1040.
Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3svjslSktQ%3D%3D&md5=6b46aee2eb8d3436ad6db108f6774a79CAS | 23465287PubMed |

Gallego, V., Martínez-Pastor, F., Mazzeo, I., Peñaranda, D. S., Herráez, M. P., Asturiano, J. F., and Pérez, L. (2013b). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in European eel (Anguilla anguilla). Aquaculture 418–419, 155–158.

Gallego, V., Violi, L., Pérez, L., Asturiano, J. F., and Yoshida, M. (2013c). Sperm quality parameters and spermatozoa morphometric characterization of marine species: an evolutionary approach from invertebrates to vertebrates. In ‘Proceedings of the 4th International Workshop on the Biology of Fish Gametes’, Albufeira, Portugal, 17–20 September 2013. (Eds S. Martínez-Páramo, C. C. V. Oliveira and M. T. Dinis.) pp. 88–89. (Centre of Marine Sciences (CCMAR), University of Algarve: Faro.)

Gibbons, B. H., Baccetti, B., and Gibbons, I. R. (1985). Live and reactivated motility in the 9 + 0 flagellum of Anguilla sperm. Cell Motil. 5, 333–350.
Live and reactivated motility in the 9 + 0 flagellum of Anguilla sperm.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL28%2Fgt1ejsQ%3D%3D&md5=b2dfdb70d0c6d7644b7746c8763b438eCAS | 2931175PubMed |

Ginneken, V. J. T. v., and Maes, G. E. (2005). The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review. Rev. Fish Biol. Fish. 15, 367–398.
The European eel (Anguilla anguilla, Linnaeus), its lifecycle, evolution and reproduction: a literature review.Crossref | GoogleScholarGoogle Scholar |

Gordon, I. R. (2004). ‘Reproductive Technologies in Farm Animals.’ (Commonwealth Agricultural Bureaux International (CABI): Wallingford, UK.)

Halpin, P. (2007). ‘Unagi: Freshwater ‘eel’. Anguilla japonica, A. anguilla, A. rostrata. Seafood Watch, Seafood Report. Technical report.’ (Monterey Aquarium: Monterey, CA, USA.)

Holt, W. V., and Harrison, R. A. P. (2002). Bicarbonate stimulation of boar sperm motility via a protein kinase A-dependent pathway: between-cell and between-ejaculate differences are not due to deficiencies in protein kinase A activation. J. Androl. 23, 557–565.
| 1:CAS:528:DC%2BD38Xlt1KhsLc%3D&md5=382cc1283ffc3ab69a9b397975d85d76CAS | 12065464PubMed |

Holt, W. V., O’Brien, J., and Abaigar, T. (2007). Applications and interpretation of computer-assisted sperm analyses and sperm sorting methods in assisted breeding and comparative research. Reprod. Fertil. Dev. 19, 709–718.
Applications and interpretation of computer-assisted sperm analyses and sperm sorting methods in assisted breeding and comparative research.Crossref | GoogleScholarGoogle Scholar | 17714625PubMed |

Hugues, J.-N. (2004). Comparative use of urinary and recombinant human chorionic gonadotropins in women. Treat. Endocrinol. 3, 371–379.
Comparative use of urinary and recombinant human chorionic gonadotropins in women.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhtFCqtLbP&md5=d98c5b378a099118832410ec21b365b7CAS | 15511131PubMed |

Kanuga, M. K., Drew, R. E., Wilson-Leedy, J. G., and Ingermann, R. L. (2012). Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss). Theriogenology 77, 916–925.
Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss).Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC383mt1CntA%3D%3D&md5=77243193f5ce026220d23a6689b6961aCAS | 22225678PubMed |

Le Comber, S. C., Faulkes, C. G., Van Look, K. J. W., Holt, W. V., and Smith, C. (2004). Recovery of sperm activity after osmotic shock in the three-spined stickleback: implications for pre-oviposition ejaculation. Behaviour 141, 1555–1569.
Recovery of sperm activity after osmotic shock in the three-spined stickleback: implications for pre-oviposition ejaculation.Crossref | GoogleScholarGoogle Scholar |

Marco-Jiménez, F., Pérez, L., de Castro, M. P. V., Garzón, D. L., Peñaranda, D. S., Vicente, J. S., Jover, M., and Asturiano, J. F. (2006). Morphometry characterisation of European eel spermatozoa with computer-assisted spermatozoa analysis and scanning electron microscopy. Theriogenology 65, 1302–1310.
Morphometry characterisation of European eel spermatozoa with computer-assisted spermatozoa analysis and scanning electron microscopy.Crossref | GoogleScholarGoogle Scholar | 16185761PubMed |

Maroto-Morales, A., Ramón, M., García-Alvarez, O., Soler, A. J., Esteso, M. C., Martínez-Pastor, F., Pérez-Guzmán, M. D., and Garde, J. J. (2010). Characterization of ram (Ovis aries) sperm head morphometry using the sperm-class analyzer. Theriogenology 73, 437–448.
Characterization of ram (Ovis aries) sperm head morphometry using the sperm-class analyzer.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3c7hs1ymsA%3D%3D&md5=1435fc41cec2fd35b8d17452fb8cfa65CAS | 20018357PubMed |

Martinez-Pastor, F., Diaz-Corujo, A. R., Anel, E., Herraez, P., Anel, L., and de Paz, P. (2005a). Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm. Theriogenology 64, 958–974.
Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm.Crossref | GoogleScholarGoogle Scholar | 16054499PubMed |

Martinez-Pastor, F., Garcia-Macias, V., Alvarez, M., Herraez, P., Anel, L., and de Paz, P. (2005b). Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process. Biol. Reprod. 72, 316–327.
Sperm subpopulations in Iberian red deer epididymal sperm and their changes through the cryopreservation process.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXptFGmtA%3D%3D&md5=fb94de5f285ca9b3b94dda5ce3e28519CAS | 15385419PubMed |

Martínez-Pastor, F., Cabrita, E., Soares, F., Anel, L., and Dinis, M. T. (2008). Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts. Reproduction 135, 449–459.
Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts.Crossref | GoogleScholarGoogle Scholar | 18367506PubMed |

Martínez-Pastor, F., Tizado, E. J., Garde, J. J., Anel, L., and de Paz, P. (2011). Statistical series: opportunities and challenges of sperm motility subpopulation analysis. Theriogenology 75, 783–795.
Statistical series: opportunities and challenges of sperm motility subpopulation analysis.Crossref | GoogleScholarGoogle Scholar | 21220164PubMed |

Miura, T., Ando, N., Miura, C., and Yamauchi, K. (2002). Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica). Zoolog. Sci. 19, 321–329.
Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica).Crossref | GoogleScholarGoogle Scholar | 12125930PubMed |

Murua, H., and Saborido-Rey, F. (2003). Female reproductive strategies of marine fish species of the North Atlantic. J. Northwest Atl. Fish. Sci. 33, 23–31.
Female reproductive strategies of marine fish species of the North Atlantic.Crossref | GoogleScholarGoogle Scholar |

Okamura, A., Yamada, Y., Horie, N., Utoh, T., Mikawa, N., Tanaka, S., and Tsukamoto, K. (2007). Effects of water temperature on early development of Japanese eel Anguilla japonica. Fish. Sci. 73, 1241–1248.
| 1:CAS:528:DC%2BD1cXjtVemtw%3D%3D&md5=61dc33eb830ea73f44a43966d8765f6aCAS |

Pankhurst, N. W., and Porter, M. J. R. (2003). Cold and dark or warm and light: variations on the theme of environmental control of reproduction. Fish Physiol. Biochem. 28, 385–389.
Cold and dark or warm and light: variations on the theme of environmental control of reproduction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXks1ajsb4%3D&md5=162dfa60828162137e2d4f0a4139f5baCAS |

Papanikolaou, E. G., Fatemi, H., Camus, M., Kyrou, D., Polyzos, N. P., Humaidan, P., Tarlatzis, B., Devroey, P., and Tournaye, H. (2010). Higher birth rate after recombinant hCG triggering compared with urinary-derived hCG in single-blastocyst IVF antagonist cycles: a randomized controlled trial. Fertil. Steril. 94, 2902–2904.
Higher birth rate after recombinant hCG triggering compared with urinary-derived hCG in single-blastocyst IVF antagonist cycles: a randomized controlled trial.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsVOktb3M&md5=b87938c73877dee9e3d6ce6b235e4d62CAS | 20580358PubMed |

Peñaranda, D. S., Marco-Jiménez, F., Pérez, L., Gallego, V., Mazzeo, I., Vicente, J. S., Jover, M., and Asturiano, J. F. (2010a). Evaluation of different diluents for short-term storage of European eel sperm under air-limited conditions. J. Appl. Ichthyology 26, 659–664.
Evaluation of different diluents for short-term storage of European eel sperm under air-limited conditions.Crossref | GoogleScholarGoogle Scholar |

Peñaranda, D. S., Pérez, L., Gallego, V., Jover, M., Tveiten, H., Baloche, S., Dufour, S., and Asturiano, J. F. (2010b). Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis. Gen. Comp. Endocrinol. 166, 160–171.
Molecular and physiological study of the artificial maturation process in European eel males: from brain to testis.Crossref | GoogleScholarGoogle Scholar | 19699741PubMed |

Pérez, L., Asturiano, J. F., Tomás, A., Zegrari, S., Barrera, R., Espinós, F. J., Navarro, J. C., and Jover, M. (2000). Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment. J. Fish Biol. 57, 1488–1504.
Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment.Crossref | GoogleScholarGoogle Scholar |

Pérez, L., Peñaranda, D., Dufour, S., Baloche, S., Palstra, A., Van Den Thillart, G., and Asturiano, J. (2011). Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females. Gen. Comp. Endocrinol. 174, 51–59.
Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females.Crossref | GoogleScholarGoogle Scholar | 21871894PubMed |

Quinn, G. P., and Keough, M. J. (2002). ‘Experimental Design and Data Analysis for Biologists.’ (Cambridge University Press: Cambridge, UK.)

R Core Team (2013). ‘R: A Language and Environment for Statistical Computing.’ (R Foundation for Statistical Computing: Vienna.)

Tanaka, H., Kagawa, H., Ohta, H., Unuma, T., and Nomura, K. (2003). The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture. Fish Physiol. Biochem. 28, 493–497.
The first production of glass eel in captivity: fish reproductive physiology facilitates great progress in aquaculture.Crossref | GoogleScholarGoogle Scholar |

Vilela, D. A. R., Silva, S. G. B., Peixoto, M. T. D., Godinho, H. P., and França, L. R. (2003). Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model. Fish Physiol. Biochem. 28, 187–190.
Spermatogenesis in teleost: insights from the Nile tilapia (Oreochromis niloticus) model.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXks1Wqt7s%3D&md5=3d0571872fbb3795443660c1c516eadbCAS |

Woolley, D. M. (1998a). Studies on the eel sperm flagellum. 2. The kinematics of normal motility. Cell Motil. Cytoskeleton 39, 233–245.
Studies on the eel sperm flagellum. 2. The kinematics of normal motility.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1c7nvVOhtA%3D%3D&md5=ead56ac6e183993746138bbd797bdc47CAS | 9519904PubMed |

Woolley, D. M. (1998b). Studies on the eel sperm flagellum. 3. Vibratile motility and rotatory bending. Cell Motil. Cytoskeleton 39, 246–255.
Studies on the eel sperm flagellum. 3. Vibratile motility and rotatory bending.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK1c7nvVOhtQ%3D%3D&md5=61e4b99bae9460f12bbbbedb2dbf5258CAS | 9519905PubMed |

Zilli, L., Schiavone, R., Chauvigné, F., Cerdà, J., Storelli, C., and Vilella, S. (2009). Evidence for the involvement of aquaporins in sperm motility activation of the teleost gilthead sea bream (Sparus aurata). Biol. Reprod. 81, 880–888.
Evidence for the involvement of aquaporins in sperm motility activation of the teleost gilthead sea bream (Sparus aurata).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlWrtr%2FE&md5=ccd60ee4afeddf00c0f7a09b34741db9CAS | 19571262PubMed |