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IN-DEPTH STUDY OF SPERM ACTIVATION IN PUFFERFISH mitochondria (located in the apix of the spermatozoon head), suggesting this cellular compartment has an important role in the activation mechanism of fish sperm. The present study showed that if there is no free Ca 2+ neither in the activation medium nor in the sperm diluent (using protocol B; with EGTA), there is no increase in [Ca 2+ ] i after activation with a non-electrolyte medium (Fig. 4B), even if motility starts. However, if any Ca 2+ remains in the activation medium and/or the sperm diluent, the spermatozoa will be able to use it, in line with its availability, and incorporate it inside the cell, thus increasing the [Ca 2+ ] i levels (Fig 4A). On the other hand, [K + ] i increased after sperm activation regardless of the media or the washing protocol used. In this respect, there are three main hypotheses about the origin/nature of the ions after sperm activation i ) the first hypothesis maintains that after activation there is an influx of Ca 2+ and/or K + through ion channels from the external environment (Morisawa, 2008); ii) the second hypothesis maintains that after activation the ions are released from intracellular stores (Morisawa, 2008); and finally iii) the last hypothesis maintains that after activation there is water efflux through specific proteins called aquoporins, and this efflux causes the increase of ion intracellular concentrations (Zilli et al., 2009, 2011). Our results suggest that the Ca 2+ ion is of an extracellular origin, due to the fact that [Ca 2+ ] i increased only when there was free calcium in the activation medium. However, these results do not agree with data previously published by Oda and Morisawa (1993), who found an increase in [Ca 2+ ] i even in the absence of this ion in the extracellular medium. However, we must taken into account the fact that in their study Ca 2+ chelator was not used in the activation medium, thus trace amounts of this ion could be masking the different results. In this respect, Krasznai et al. (2000) showed that levels of [Ca 2+ ] i in fish spermatozoa seminal plasma are particularly low (40-70 nM) and negligible remains of extracellular Ca 2+ could be enough to interact with spermatozoa. Regarding the K + ion, our data show that [K + ] i increased regardless of the composition of the activation media and therefore, the K + ion would be of an intracellular origin. However, intracellular potassium stores in animal cells have not been proven, so more studies may be necessary to determine the origin/nature of this ion in marine fish sperm. 103
CHAPTER 4 5. Conclusions Some conclusions regarding different issues of pufferfish sperm have emerged from this study: i) Through the use of diluents, it is possible to preserve pufferfish sperm for a short-term period (up to 7 days without differences compared to fresh sperm) for use in aquaculture matters. ii) Medium osmolality is the most important factor in triggering trigger sperm motility, and values of around 750-825 mOsm/kg are necessary to activate this process in pufferfish, which seems to be a non-dosedependent mechanism in terms of osmolality. iii) The ion composition of the activation media is able to modulate the sperm activation process. Despite spermatozoa being able to initiate movement without any ion in the activation medium, the presence/absence of these ions can affect the kinetic parameters of spermatozoa. iv) In the natural environment, the activation of sperm motility generates an increase in intracellular Ca 2+ and K + , suggesting these ions have an important role in the activation mechanism of marine fish sperm. Acknowledgements Funded by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES- 2009-020310) and has been granted a fellowship (EEBB-I-12-05858) from the Spanish Personnel Research Training Programme to carry out this study in the Misaki Marine Biological Station (Miura, Japan). 104
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SPERM PHYSIOLOGY AND QUALITY IN TWO
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ACKNOWLEDGEMENTS Muchos años han p
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aunque tiene una alcachofa en la ca
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TABLE OF CONTENTS SUMMARY 1 RESUMEN
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SUMMARY SUMMARY The conservation st
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RESUMEN RESUMEN Las especies objeto
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RESUM RESUM Les espècies objecte d
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GENERAL INTRODUCTION 7
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GENERAL INTRODUCTION who postulated
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GENERAL INTRODUCTION 2. Sperm physi
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GENERAL INTRODUCTION Finally, the a
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GENERAL INTRODUCTION 3. Sperm quali
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GENERAL INTRODUCTION in fish (Kime
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GENERAL INTRODUCTION However, for s
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GENERAL INTRODUCTION future applica
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GENERAL INTRODUCTION With regards t
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OBJECTIVES The chapters presented b
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STANDARDIZATION OF SPERM MOTILITY E
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CHAPTER 2 Study of the effects of t
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CHAPTER 2 reducing the pressure on
Page 61 and 62: CHAPTER 2 ongoing task in order to
Page 63 and 64: CHAPTER 2 To measure sperm density,
Page 65 and 66: CHAPTER 2 Spermiating males (%) 100
Page 67 and 68: CHAPTER 2 100 80 60 I II III IV A 4
Page 69 and 70: CHAPTER 2 3.2 Hormonal treatments P
Page 71 and 72: CHAPTER 2 With regard to the sperm
Page 73 and 74: CHAPTER 2 Relative volume (%) 80 I
Page 75 and 76: CHAPTER 2 In this respect, it is we
Page 77 and 78: CHAPTER 2 treatment to that of the
Page 79 and 80: CHAPTER 2 Our results demonstrate t
Page 82 and 83: ROLE OF IONS IN THE SPERM ACTIVATIO
Page 92: CHAPTER 4 Study of pufferfish (Taki
Page 95 and 96: CHAPTER 4 2002) so Takifugu niphobl
Page 97 and 98: CHAPTER 4 carrying out sperm collec
Page 99 and 100: CHAPTER 4 Table 1. Activation media
Page 101 and 102: CHAPTER 4 TM (%) 100 A a a Und-PD a
Page 103 and 104: CHAPTER 4 TM (%) 80 60 40 20 0 A a
Page 105 and 106: CHAPTER 4 TM (%) 80 A ASW100 GLU 60
Page 107 and 108: CHAPTER 4 although the activation m
Page 109 and 110: CHAPTER 4 dilution ratio and an opt
Page 111: CHAPTER 4 2006). Several studies in
Page 116 and 117: RELATIONSHIP BETWEEN SPERM MOTILIY
Page 130: RELATIONSHIP BETWEEN SPERM MOTILIY
Page 134 and 135: GENERAL DISCUSSION 1. Improvements
Page 136 and 137: GENERAL DISCUSSION profiles induced
Page 138 and 139: GENERAL DISCUSSION more studies on
Page 140 and 141: GENERAL DISCUSSION sperm motility p
Page 142: CONCLUSIONS 133
Page 145 and 146: CONCLUSIONS vii. The coefficients o
Page 148 and 149: REFERENCES Aarestrup K, Økland F,
Page 150 and 151: REFERENCES Beirão J, Cabrita E, P
Page 152 and 153: REFERENCES Cabrita E, Robles V, Cu
Page 154 and 155: REFERENCES De Vos A, Van De Velde H
Page 156 and 157: REFERENCES Gallego V, Pérez L, Ast
Page 158 and 159: REFERENCES sperm competition and fe
Page 160 and 161: 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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