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RELATIONSHIP BETWEEN SPERM MOTILIY AND FERTILIZATION RATES Figure 1. Schematic diagram of some kinetic parameters recorded by CASA system. Black circles represent successive positions of the head of a motile sperm through the video recording. Sperm movement parameters: VCL, curvilinear velocity; VAP, averaged path velocity; VSL, straight-line velocity; ALH, amplitude of lateral head displacement; BCF, beat/cross frequency. 2.3 Experimental setup: fertilization and hatching trials. This study was divided into two trials as described in Fig. 2. In Trial 1, gametes from five males and two females were separately used for fertilizations assays, using 5 of the 10 possible combinations (1 male, 1 female), which were simultaneously assayed in three different sperm/egg ratios (10 3 , 10 4 and 10 5 spermatozoa/egg) and using sperm activated after three different times (5, 20 and 40 s). TRIAL 1 TRIAL 2 Sperm/egg (10 3 ) Sperm/egg (10 4 ) Sperm/egg (10 5 ) Sperm/egg (10 4 ) 5s 20s 40s 5s 20s 40s 5s 20s 40s 5s 20s 35s M1xF1 M1xF1 M1xF1 M1xF1 M1xF1 M1xF1 M1xF1 M1xF1 M1xF1 M6xF3 M6xF3 M6xF3 M2xF1 M2xF1 M2xF1 M2xF1 M2xF1 M2xF1 M2xF1 M2xF1 M2xF1 M7xF3 M7xF3 M7xF3 M3xF1 M3xF1 M3xF1 M3xF1 M3xF1 M3xF1 M3xF1 M3xF1 M3xF1 M8xF3 M8xF3 M8xF3 M4xF2 M4xF2 M4xF2 M4xF2 M4xF2 M4xF2 M4xF2 M4xF2 M4xF2 M9xF3 M9xF3 M9xF3 M5xF2 M5xF2 M5xF2 M5xF2 M5xF2 M5xF2 M5xF2 M5xF2 M5xF2 M10xF33 M10xF3 M10xF3 Figure 2. Experimental crosses for Trial 1 and 2 (n=15). M: male; F: female. 111
CHAPTER 5 Eggs from two females were divided into batches of 80-90 eggs and placed into 60 × 15 mm Petri dishes using a micropipette with the tip cut off to prevent compression of the eggs. A known aliquot of sperm (adjusting the volume according to the calculated sperm/egg ratio) was added to 5 ml of seawater and then the sperm-water solution was added to the corresponding batch of eggs at different post-activation times. After an incubation period of 10 min, the eggs were transferred into a clean Petri dish for incubation with 8 mL of clean seawater. The eggs were then incubated in darkness at a controlled temperature of 20 °C. Fertilization rates were evaluated between 1-2 hrs after insemination by counting the percentage of embryos which reached the 4-cell stage in relation to the total number of eggs used. In Trial 2, using the 104 sperm/egg ratio, new batches of 80-90 eggs from one female were separately fertilized with sperm from five males at different post-activation times (5, 20 and 35 s) with the goal of establishing correlations between sperm quality parameters and fertilization and hatching rates. The gamete collection and the artificial insemination were carried out in the same manner as in Trial 1 and the hatching rates were calculated as the percentage of hatched larvae in relation to the total number of eggs. Dead eggs and larvae were removed and counted when detected during daily inspections, and seawater was exchanged once a day. 2.4 Statistical analysis The mean and standard error were calculated for all the sperm quality parameters. Shapiro-Wilk and Levene tests were used to check the normality of data distribution and variance homogeneity, respectively. Oneway analysis of variance (ANOVA) was used to analyse the data. Significant differences were detected using the Tukey´s multiple range test (P < 0.05). Pearson´s correlation, coefficient of determination and linear regression analysis were used to find the relationship between the different sperm quality parameters and fertilization/hatching rates. All statistical analyses were performed using the statistical package SPSS version 19.0 for Windows software (SPSS Inc., Chicago, IL, USA). 112
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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
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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
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
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
Page 162 and 163: REFERENCES Mortimer ST. Effect of i
Page 164 and 165: REFERENCES Peñaranda DS, Marco-Jim
Page 166 and 167: REFERENCES Rurangwa E, Volckaert FA
Page 168 and 169: REFERENCES Tesch F. Telemetric obse
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REFERENCES Wong PYD, Lee WM, Tsang
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