Universidad de Len
Facultad de C.C. Biolgicas y Ambientales
Dpto. Biologa Molecular
rea Biologa Celular
Sperm quality in marine teleosts: applications to broodstock management and sperm storage in two farmed species Solea senegalensis and
Sparus aurata Calidad del semen en telesteos marinos: aplicaciones a la gestin de reproductores y al almacenamiento del semen de dos especies
cultivadas Solea senegalensis y Sparus aurata
By
Jos Beiro Santos
Thesis presented for the obtention of PhD grade
Memoria presentada como parte de los requerimientos para optar al ttulo de Doctor
Len, 30 de Mayo de 2011
Jos Beiro received a PhD grant SFRH / BD / 31990 / 2006
financed by the POPH QREN (Tipologia 4.1)
and co-funded by the European Social Fund
and by national funds from the
Portuguese Ministry of Science and Technology.
Uma mosca sem valor pousa com a
mesma alegria na careca de um doutor
como em qualquer porcaria
Antnio Aleixo (1899-1949)
CCoonntteennttss // nnddiiccee
Summary ......................................................................................................................................... 7
Introduction ............................................................................................................................... 21
The importance of control reproduction for aquaculture and its limitations .......................... 23
Sperm quality ............................................................................................................................ 24
Basic quality parameters ....................................................................................................... 25
Morphology ........................................................................................................................... 25
Sperm motility ........................................................................................................................ 25
Motility activation ................................................................................................................. 26
Motility measurement ........................................................................................................... 26
ATP ......................................................................................................................................... 28
Mitochondria ......................................................................................................................... 28
Metabolism ............................................................................................................................ 29
Viability .................................................................................................................................. 29
DNA ........................................................................................................................................ 30
Apoptosis ............................................................................................................................... 30
Seminal plasma composition ................................................................................................. 31
Sperm membrane composition ................................................................................................ 31
Factors affecting sperm quality ................................................................................................. 33
Nutrition ................................................................................................................................. 33
Husbandry conditions and abiotic/biotic factors .................................................................. 35
Hormonal treatments ............................................................................................................ 36
Broodstock selection .............................................................................................................. 36
The use of sperm cryopreservation to overcome some reproductive constraints... ............ 37
Improving cryopreservation protocols ................................................................................... 37
The effect of cryopreservation on plasma membranes ......................................................... 40
Solea Senegalensis - a promising species to aquaculture with reproductive constraints ....... 41
Sparus aurata - an excellent aquaculture model for sperm cryopreservation studies of marine teleosts ..................................................................................................................................... 42
References ................................................................................................................................. 43
Objectives ..................................................................................................................................... 53
Chapter 1 WILD CAPTIVE SOLEA SENEGALENSIS SPERM QUALITY ANALYSIS AND IMPROVEMENT ............................................................................................................................ 57
1.1.Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level ............................................................................................................................................... 59
Abstract ..................................................................................................................................... 63
1.Introduction ........................................................................................................................... 64
2.Material and Methods ........................................................................................................... 65
2.1.Chemicals ......................................................................................................................... 65
2.2.Broodstock husbandry conditions and sampling ............................................................. 65
2.3.Sperm quality analysis ..................................................................................................... 66
2.4.Statistical analysis ........................................................................................................... 68
3.Results .................................................................................................................................... 68
3.1.Motility ............................................................................................................................ 68
3.2.Hyperosmotic test ............................................................................................................ 71
3.3.Apoptosis assay ............................................................................................................... 72
3.4.DNA integrity ................................................................................................................... 74
4.Discussion ............................................................................................................................... 75
Acknowledgements ................................................................................................................... 78
References ................................................................................................................................. 79
1.2.Changes in Solea senegalensis sperm quality throughout the year .................................... 83
Abstract ..................................................................................................................................... 87
1.Introduction ........................................................................................................................... 88
2.Material and Methods ........................................................................................................... 89
2.1.Broodstock husbandry conditions ................................................................................... 89
2.2.Sampling .......................................................................................................................... 89
2.3.Sperm analysis ................................................................................................................. 90
2.4.Statistical analysis ........................................................................................................... 91
3.Results .................................................................................................................................... 91
3.1.Sperm production and motility evaluation ...................................................................... 91
3.2.DNA fragmentation and apoptosis .................................................................................. 93
3.3.Sperm membrane resistance to seawater ....................................................................... 94
3.4.Correlations between the sperm parameters .................................................................. 95
4.Discussion .............................................................................................................................
