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IN-DEPTH STUDY OF SPERM ACTIVATION IN PUFFERFISH 4. Discussion 4.1 Short-term storage With a view to seeking a way to improve the handling of fish sperm used for aquaculture, ecological (repopulations) or scientific purposes, several chilled storage methods have been developed to preserve sperm integrity and quality over time. However, several factors such as the incubation temperature, the composition of the diluent, the dilution ratio or environmental conditions should be taken into account when designing a proper storage protocol (Peñaranda et al., 2010c,d). The first step of this process involves deciding whether the sperm will be preserved undiluted or, on the contrary, diluted in a medium. It has been reported that fish sperm samples stored undiluted tend to show poorer motility values than diluted sperm samples (Babiak et al., 2006; DeGraaf and Berlinsky, 2004; Peñaranda et al., 2010c). Our results agree, with undiluted pufferfish sperm showing significantly lower values in the sperm quality parameters than diluted sperm samples at all the incubation times. On the other hand, within the undiluted samples, microtube vials generated better results than PD storage. This could be explained at least in part by the effect of the aerobic and anaerobic spermatozoa pathway. While the spermatozoa stored in microtube would have been subjected to an atmosphere with poor oxygen levels due to the shape of the vial, thus inducing the use of the anaerobic pathway; the spermatozoa stored in Petri dishes would have been in a richer oxygen atmosphere, thus using the aerobic pathway. It has been reported that aerobic pathway produce higher levels of oxygen free radicals (ROS, Kowalowka et al., 2007), which have been associated with defective sperm function in fish spermatozoa (Bansal and Bilaspuri, 2010; Martínez-Páramo et al., 2012; Pérez-Cerezales et al., 2009). Therefore, the use of the aerobic pathway of undiluted spermatozoa stored in Petri dishes would generate a higher level of ROS and thus, a sharp decrease in sperm quality parameters. On the other hand, the second step to designing an optimum short-term storage method involves selecting a suitable diluent medium, with a proper 99
CHAPTER 4 dilution ratio and an optimum incubation temperature. Seminal plasma in almost all teleost fish is composed of a mix of ions including Na + , Ca 2+ , K + , Cl - , etc., with an osmolality of 300-350 mOsm⁄kg (Asturiano et al., 2004b; Morisawa, 2001, 2008; Pérez et al., 2003). In this respect, we used the common dilution medium used in pufferfish sperm studies (Krasznai et al., 2003; Takai and Morisawa, 1995) applying a dilution ratio of 1:50 (v/v), which had been tested in other fish species with good results (Ohta and Izawa, 1996; Peñaranda et al., 2010d). In the present trial, the first significant differences between the diluted samples and the fresh samples were found after 7 days of incubation in the velocity parameters and, after 11 days, all diluted samples showed lower motility values than fresh samples. In this respect, the diluent acted by prolonging the quality of stored spermatozoa, providing better control of the physicochemical conditions during storage through avoiding negative effects such as desiccation, contamination and unbalanced gas exchange (Babiak et al., 2006). It is worth highlighting this result as it allows the preservation and use of pufferfish sperm during a short-term period for aquaculture matters. Regarding temperature, it has been reported that low temperatures result in better motilities than high temperatures for ATP-spending reasons (Alavi and Cosson, 2005), thus a temperature of 4 ºC was used in this trial. Finally, in addition to the choice of diluent and its dilution ratio, there is the possibility of adding some reagents such as membrane protectors to the medium. In this respect, bovine serum albumin (BSA) has been used with good results in other species such as gilthead seabream (Cabrita et al., 2005b), European sea bass (Zilli et al., 2003) and European eel (Peñaranda et al., 2010c). However, no differences were registered between the diluting media with or without BSA in our experiment, therefore we do not recommend the use of this reagent in the chilled storage of pufferfish sperm because it can increase the chances of bacterial growth. 4.2 Effect of the osmolality on sperm quality parameters In the natural environment seawater has an osmolality of 1000-1100 mOsm/kg, with a high variety of ions. However, in order to find out the 100
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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
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: CHAPTER 4 although the activation m
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
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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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