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SUMMARY SUMMARY The conservation status of the species studied in this thesis (European eel and pufferfish) is currently frail, thus the main goal of this research was to develop, improve and apply several techniques and protocols with the aim of increasing the knowledge about their sperm biology, improving their reproductive performance and even helping future breeding in captivity. The reproductive performance of the males is often assessed through the sperm motion parameters analysed by the CASA system, so first we focused on how to standardize this technique in terms of procedural and biological settings. In this respect, we laid the foundations for applying a standard method to assess sperm quality in fish, making it possible for sperm studies to be compared both intra- and inter-laboratories using the proper CASA settings. Secondly, with the aim of improving the reproductive performance of European eel males, 3 thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and 3 hormonal treatments (hCG, hCG rec and PSMG) were assessed based on different sperm quality parameters. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced by water temperature, with treatment T20 showing the best results in all the sperm quality parameters. In the case of hormonal treatments, hCG rec produced the best results in all the sperm quality parameters, becoming an economically profitable/viable treatment and an effective alternative to the standard hCG treatment often used to induce spermiation in eel species. A preliminary physiological study regarding the changes to the main ions involved in the fish sperm activation process was carried out. Our results showed that intracellular concentrations of Ca 2+ and K + increased upon eel sperm activation, while pH gradually decreased over time, thus it is likely that all of them play an important role in the initiation of sperm motility in the European eel, as with other marine and freshwater teleosts. 1
SUMMARY In the second part of this thesis, which focuses on the pufferfish, an indepth study into the sperm of this species was carried out for future application in aquacultural matters. A short-term storage method for pufferfish sperm was developed, enabling us to preserve the sperm quality parameters for a relatively long time period (7 days) compared to fresh sperm samples. Moreover, the effects of both the osmolality and the ion composition of the activation media on the sperm motion parameters were evaluated, concluding that both factors play an essential role in the initiation of sperm motility of pufferfish sperm and probably, in marine fish sperm. Finally, in vitro fertilization trials were developed to assertain how the quantity and quality of male gametes affects the fertilization and hatching rates. We demonstrated that sperm/egg ratio and sperm quality are strongly related factors, suggesting that both should be taken into account as unique interrelated elements. In addition, coefficients of correlation among all the spermatozoa motion parameters provided by a CASA system and fertilization and hatching rates were estimated for the first time in a marine species. Spermatozoa velocities showed the highest coefficients (≥0.80), suggesting that the kinetics of the spermatozoa are a key factor in the fertilization process. 2
Page 2 and 3: SPERM PHYSIOLOGY AND QUALITY IN TWO
Page 4 and 5: ACKNOWLEDGEMENTS Muchos años han p
Page 6 and 7: aunque tiene una alcachofa en la ca
Page 8 and 9: TABLE OF CONTENTS SUMMARY 1 RESUMEN
Page 12 and 13: RESUMEN RESUMEN Las especies objeto
Page 14 and 15: RESUM RESUM Les espècies objecte d
Page 16: GENERAL INTRODUCTION 7
Page 19 and 20: GENERAL INTRODUCTION who postulated
Page 21 and 22: GENERAL INTRODUCTION 2. Sperm physi
Page 23 and 24: GENERAL INTRODUCTION Finally, the a
Page 25 and 26: GENERAL INTRODUCTION 3. Sperm quali
Page 27 and 28: GENERAL INTRODUCTION in fish (Kime
Page 29 and 30: GENERAL INTRODUCTION However, for s
Page 31 and 32: GENERAL INTRODUCTION future applica
Page 33 and 34: GENERAL INTRODUCTION With regards t
Page 36: OBJECTIVES The chapters presented b
Page 40 and 41: STANDARDIZATION OF SPERM MOTILITY E
Page 56: CHAPTER 2 Study of the effects of t
Page 59 and 60: CHAPTER 2 reducing the pressure on
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CHAPTER 2 ongoing task in order to
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CHAPTER 2 To measure sperm density,
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CHAPTER 2 Spermiating males (%) 100
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CHAPTER 2 100 80 60 I II III IV A 4
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CHAPTER 2 3.2 Hormonal treatments P
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CHAPTER 2 With regard to the sperm
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CHAPTER 2 Relative volume (%) 80 I
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CHAPTER 2 In this respect, it is we
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CHAPTER 2 treatment to that of the
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CHAPTER 2 Our results demonstrate t
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ROLE OF IONS IN THE SPERM ACTIVATIO
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CHAPTER 4 Study of pufferfish (Taki
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CHAPTER 4 2002) so Takifugu niphobl
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CHAPTER 4 carrying out sperm collec
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CHAPTER 4 Table 1. Activation media
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CHAPTER 4 TM (%) 100 A a a Und-PD a
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CHAPTER 4 TM (%) 80 60 40 20 0 A a
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CHAPTER 4 TM (%) 80 A ASW100 GLU 60
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CHAPTER 4 although the activation m
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CHAPTER 4 dilution ratio and an opt
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CHAPTER 4 2006). Several studies in
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CHAPTER 4 5. Conclusions Some concl
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RELATIONSHIP BETWEEN SPERM MOTILIY
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GENERAL DISCUSSION 1. Improvements
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GENERAL DISCUSSION profiles induced
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GENERAL DISCUSSION more studies on
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GENERAL DISCUSSION sperm motility p
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CONCLUSIONS 133
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CONCLUSIONS vii. The coefficients o
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REFERENCES Aarestrup K, Økland F,
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REFERENCES Beirão J, Cabrita E, P
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REFERENCES Cabrita E, Robles V, Cu
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REFERENCES De Vos A, Van De Velde H
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REFERENCES Gallego V, Pérez L, Ast
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REFERENCES sperm competition and fe
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REFERENCES Liu QH, Li J, Xiao ZZ, D
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REFERENCES Mortimer ST. Effect of i
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REFERENCES Peñaranda DS, Marco-Jim
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REFERENCES Rurangwa E, Volckaert FA
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REFERENCES Tesch F. Telemetric obse
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REFERENCES Wong PYD, Lee WM, Tsang
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