chapter 3 - RiuNet

More documents

Recommendations

Info

ROLE OF IONS IN THE SPERM ACTIVATION were less than 0.05 % in all the cases, and therefore a DMSO effect on motility could be discarded. 2.3.2 Determination of intracellular Ca 2+ and K + and pH After the incubation time, 5 µl of sperm sample was added to a tube containing P1 medium (500 µl) to measure the fluorescence emitted by the specific ion in the quiescent stage. Later, 5 µl of sperm sample was added to a tube containing activation medium (seawater, 500 µl) and the fluorescence emitted by sperm cells at 30, 60 and 120 s after activation was recorded. Fluo-4 AM and Snarf-5F AM were both excited by the blue laser (488 nm), and their fluorescence was read with the FL1 (530/40BP filter) and FL4 (680/30BP filter) photodetector, respectively. PBFI AM was excited by ultraviolet light (340 nm) and its fluorescence was read with the FL6 photodetector (450/50BP filter). TO-PRO-3 was excited by the use of red laser (635 nm), and its red fluorescence was read with the FL8 photodetector (665/20BP filter). The fluorescence data was displayed in logarithmic mode. Ten thousand events were collected per sample, with a flow rate of 200 cells/s, using a gate in forward and side scatter to exclude debris and aggregates from the analysis. 2.4 Location of Ca 2+ and K + in quiescent spermatozoa An aliquot of sperm samples incubated with Fluo-4 AM or PBFI AM (see section 2.3.1) was used to obtain microphotographs through a Nikon Eclipse E600 microscope. UV-2A (ultraviolet excitation and blue emission) and B-2A (blue excitation and green emission) filters were used to obtain Ca 2+ and K + images, respectively. 2.5 Statistical analysis Weasel software (WEHI, Victoria, Australia) was used to analyze the data obtained by flow cytometry. After removing dead spermatozoa (TO-PRO®- 3) from the analysis, the mean fluorescence intensity (MFI, arbitrary units) was obtained from each sample. Statistical analyses were performed using 77
CHAPTER 3 the statistical package SPSS version 19.0 for Windows software (SPSS Inc., Chicago, IL, USA). 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 analyze data with normal distribution. Significant differences were detected using the Tukey multiple range test (P
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 10 and 11:
SUMMARY SUMMARY The conservation st
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
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
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
Page 170 and 171:
REFERENCES Wong PYD, Lee WM, Tsang
show all

chapter 3 - RiuNet

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?