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STANDARDIZATION OF SPERM MOTILITY EVALUATION Abstract The development of powerful computerized-assisted sperm analysis software (CASA) has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rates, sperm dilution, chamber models, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to measure sperm quality parameters on the European eel correctly. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10x was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity, we therefore recommend using the camera's high frame rate setting available. Finally, the reduction in sperm motility over post-activation times suggests that sperm analysis should be performed within the first 60 s after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by CASA software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, as marine fish species show relatively similar features to sperm kinetic parameters, these results could be considered for the evaluation of sperm motility in other fish species. 1. Introduction The economic importance and high commercial demand of the European eel, Anguilla anguilla, primarily from European and Japanese markets, is well known (Dekker, 2000; Moriarty and Dekker, 1997; Pérez et al., 2004). However, the population of the European eel has declined to such a degree that major concerns have been raised for its long-term survival (Feunteun, 2002; Stone, 2003). Efforts have been made to understand the life cycle and reproductive biology of this species (Tesch, 1978; van Ginneken and Maes, 31
CHAPTER 1 2005) and we already know it is necessary to use hormones to induce ovulation and spermiation to overcome the lack of normal spawning stimuli in captivity (Asturiano et al., 2005; Pérez et al., 2008). It is particularly advantageous to stimulate the spermiation of male eels so that sperm is available in a short time and in high volume (Gallego et al., 2012). In this respect, knowledge of how to manipulate and preserve eel sperm is essential (Peñaranda et al., 2008, 2009 and 2010) and a reliable and standardized method to analyse its quality is needed. The evaluation of sperm motility and other kinetic parameters like curvilinear, straight line, and average path velocities, and morphology, is an essential tool in the examination of sperm quality in many fish species (Liu et al., 2007; Marco-Jiménez et al., 2008; Gallego el at., 2012), including the European eel (Gallego el at., 2012; Marco-Jiménez et al., 2006; Asturiano et al., 2007). Despite the fact that for many years optic microscopes have conventionally been used to carry out analyses and evaluations, it is considered subjective method and great variations have been reported (Coetzee et al., 1999). According to Verstegen et al. (2002), when subjective optical microscopic evaluation is used in humans and animals, variations of 30% to 60% have been reported in the estimation of the motility parameters of the same ejaculates. The computer assisted sperm analysis, or CASA, has been used by an increasing number of researchers worldwide and provides an objective, rapid, and multiple-parameter assessment of sperm quality. To make it possible to compare the results obtained by different laboratories, all studies that use CASA must describe its methodology very clearly, particularly concerning image acquisition rate, track sampling time, number of cells sampled, type and depth of the chamber used, software name, microscope optic and magnification, etc. (Castellini et al., 2011; Soler et al., 2012; Rosenthal et al., 2009). Unfortunately, in most publications, details of these parameters are not provided, thus reducing the possibility of comparing the results of different laboratories. Furthermore, because there are many different configurations and methods of using CASA, it is important to establish standard methods of enhancing the reliability, 32
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 42 and 43: 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
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ROLE OF IONS IN THE SPERM ACTIVATIO
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CHAPTER 4 Study of pufferfish (Taki
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CHAPTER 4 2002) so Takifugu niphobl
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CHAPTER 4 carrying out sperm collec
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CHAPTER 4 Table 1. Activation media
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CHAPTER 4 TM (%) 100 A a a Und-PD a
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CHAPTER 4 TM (%) 80 60 40 20 0 A a
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CHAPTER 4 TM (%) 80 A ASW100 GLU 60
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CHAPTER 4 although the activation m
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CHAPTER 4 dilution ratio and an opt
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CHAPTER 4 2006). Several studies in
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CHAPTER 4 5. Conclusions Some concl
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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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