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ROLE OF IONS IN THE SPERM ACTIVATION motility inhibition, that was not observed when sperm was maintained in an extender at physiological pH (8.5) (Peñaranda et al., 2009). Thus, this study was designed to observe the changes in intracellular Ca 2+ , K + , and pH after motility activation in European eel spermatozoa. In this study we used flow cytometry to describe the variations of these factors from the immotile stage to the motile stage, at different times after the initiation of sperm motility, with the aim of establishing the first hypothesis on motility activation in this species. 2. Materials and methods 2.1 Fish handling and sampling Fifteen adult eel males (100±2 g; 40±5 cm) from the fish farm Valenciana de Acuicultura, S.A. (Puzol, Valencia; East coast of Spain) were moved to the aquaculture facilities at the Universidad de León (Spain). The fish were distributed in three 60-L aquaria (5 males per aquarium) equipped with separate recirculation systems, thermostats and covered to maintain constant darkness. The eels were gradually acclimatized to artificial seawater (Aqua Medic Meersalz, 37 g/l) and once a week they received an intraperitoneal injection of hCG (1.5 IU g -1 fish; Argent Chemical Laboratories, USA), after being anesthetized (benzocaine, 60 ppm) and individually weighed. The total treatment lasted 14 weeks, and sperm samples were obtained from the sixth week until the end of the treatment. The fish were fasted throughout the experiment and were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 86/609/EEC). 2.2 Sperm collection and evaluation of motility Sperm samples were collected 24 h after the administration of the hormone to obtain the highest quality sperm (Pérez et al., 2000). The eels were anesthetized, and the genital area was first cleaned with distilled water and then thoroughly dried to avoid contaminating the sperm with faeces, urine or seawater. Then, the sperm was collected by applying gentle 75
CHAPTER 3 abdominal pressure with the help of a small modified aquarium air pump which produced a vacuum breathing force to help collect the sperm in plastic Falcon tubes. Sperm samples were kept at 4 ºC until the motility analyses, which were performed less than 1 hour after collection. Sperm motility was assessed subjectively in triplicate by a trained observer, after mixing 1 µl of sperm with 200 µl of artificial seawater (SW; Aqua Medic Meersalz, 37 g/l, with 2% BSA (w/v), pH adjusted to 8.2) and observed under a microscope in a glass slide. Samples having >50% of motile cells were selected to study intracellular ionic changes. They were diluted 1:100 in P1 medium (in mM: NaCl 125, NaHCO 3 20, KCl 30, MgCl 2 2.5, CaCl 2 1, and pH 8.5; Peñaranda et al., 2010) and maintained at 4 ºC until the flow cytometry analysis. 2.3 Flow citometry The relative amounts of different ions were determined by flow cytometry using a CyAn ADP Flow Cytometer (Beckman Coulter, Brea, CA) equipped with an argon ion laser. Slightly angled scattered front light was used for the electronic gating of data collection, allowing us to exclude dead cells from the analyses. 2.3.1 Incubation protocol For Ca 2+ , a stock solution of 1mM Fluo-4 AM (Invitrogen F14201) in DMSO was kept at -20 ºC until use. A sample of 100 µl diluted sperm was incubated with 0.5 µl Fluo-4 AM (final concentration 5 µM) at RT (20 ºC) for 30 minutes. For K + , a stock solution of 1mM PBFI AM (Invitrogen, P1267) in DMSO was kept at -20 ºC until use. A sample of 100 µl diluted sperm was incubated with 0.5 µl PBFI AM (final concentration 5 µM) at RT (20 ºC) for 90 minutes. For pH i , a stock solution of 1mM Snarf-5F AM (Invitrogen, S23923) in DMSO was kept at -20 ºC until use. A sample of 100 µl diluted sperm was incubated with 0.5 µl Snarf-5F AM (final concentration 5 µM) at RT (20 ºC) for 45 minutes.To exclude dead cells from the analysis, the spermatozoa were also incubated with TO-PRO®-3 (Invitrogen T7596) to reach a final concentration of 2 µM. Final DMSO concentrations in sperm 76
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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
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
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 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
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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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