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IN-DEPTH STUDY OF SPERM ACTIVATION IN PUFFERFISH In addition, different results can be found in literature for several species. In the case of seawater tilapia (Oreochromis mossambicus), it has been reported that extracellular Ca 2+ as well as osmotic pressure are both essential factors for sperm activation (Linhart et al., 1999); however, Krasznai et al. (2003) showed that extracellular Ca 2+ was not necessary for sperm activation in pufferfish, but rather a hyperosmotic shock is required to release Ca 2+ from the intracellular stores; in the case of the European eel it has been reported that intracellular Ca 2+ and K + ions increase upon activation, and may have an important role in the initiation of spermatozoa motility; and finally, in the case of Atlantic croaker (Micropogonias undulatus), in addition to Ca 2+ and K + , Na + and Cl - ions seem to be involved in sperm activation (Detweiler and Thomas, 1998). Thus, the second goal of this study was to evaluate the effects of environmental factors, including the composition and the osmolality of the medium, on the motility characteristics of spermatozoa, as well as to measure the intracellular concentrations of the main ions involved in sperm activation in pufferfish. 2. Materials and methods 2.1 Fish handling, sperm collection and sampling The pufferfish displays a unique spawning behaviour at Arai Beach near the Misaki Marine Biological Station (MMBS, Japan). Large schools of fish arrive to the beach with the spring tide around the new moon during the spawning season between June and July (Yamahira, 1996). Spawning takes place repeatedly from 2 hours before the sunset to that moment, and during that time, pufferfish males were caught and moved to the MMBS facilities. Fish were kept in running seawater tanks at 18 °C and were fed a commercial pellet throughout the experiment. All the experiments were carried out in compliance with the animal guidelines of the University of Tokyo on Animal Care. Fish in captivity were able to produce sperm several days after stocking and sperm samples were collected periodically over the sampling days. Before 87
CHAPTER 4 carrying out sperm collection the genital area was cleaned with freshwater and thoroughly dried to avoid contamination of the samples with faeces, urine or seawater. Sperm samples (approximately 1 mL) were collected by applying gentle pressure to the fish abdomen, they were then maintained at 4 ºC until analysis and evaluated in the first hour after extraction. 2.2 Evaluation of motility and kinetic sperm parameters Sperm was diluted (1:50) before the activation process in seminal plasmalike solution (SLS) consisting in 130 mM NaCl, 5 mM KCl, 10 mM HEPES and 1 mM CaCl 2 , pH adjusted to 7.5 (Krasznai et al., 2003). Sperm was activated by mixing 0.5 µl of this dilution with 4 µl of artificial seawater (ASW100) comprised of 460 mM NaCl, 10 mM KCl, 36 mM MgCl 2 , 17 mM MgSO 4 , 9 mM CaCl 2 , and 10 mM HEPES, with 1% BSA (w/v) and pH adjusted to 8.2. The sperm-seawater mix was put in a SpermTrack-10® chamber and observed (Proiser R+D, S.L.; Paterna, Spain). Video sequences were recorded through a high-sensitivity video camera (HAS-220; 50 fps) mounted on a phase contrast microscope (Olympus BX51) with a 10x objective lens (Olympus Splan NH). All the motility analyses were performed by triplicate using the motility module of ISAS (Proiser R+D, S.L.; Paterna, Spain). The parameters considered in this study were total motility (TM, %), defined as the percentage of motile cells; progressive motility (PM, %), defined as the percentage of spermatozoa which swim in a essentially straight line; curvilinear velocity (VCL, µm/s), defined as the time/average velocity of a sperm head along its actual curvilinear trajectory; straight line velocity (VSL, µm/s), defined as the time/average velocity of a sperm head along the straight line between its first detected position and its last position; average path velocity (VAP, µm/s), defined as the time/average of sperm head along its average spatial trajectory; the percentage of fast (FA; VAP > 100 µm/s), medium (ME; VAP = 50-100 µm/s) and slow (SL; VAP = 10- 50 µm/s) spermatozoa; straightness (STR, %), defined as the linearity of the average spatial path; linearity (LIN, %), defined as the linearity of the curvilinear trajectory; wobble (WOB, %), defined as the trajectory 88
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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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Page 46 and 47: 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
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: CHAPTER 4 2002) so Takifugu niphobl
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 and 112: CHAPTER 4 2006). Several studies in
Page 113 and 114: CHAPTER 4 5. Conclusions Some concl
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
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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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