Table Of ContentMethods in
Reproductive
Aquaculture
Marine and Freshwater Species
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Marine Biology
SERIES
The late Peter L. Lutz, Founding Editor
David H. Evans, Series Editor
PUBLISHED TITLES
Biology of Marine Birds
E.A. Schreiber and Joanna Burger
Biology of the Spotted Seatrout
Stephen A. Bortone
The Biology of Sea Turtles, Volume II
Peter L. Lutz, John A. Musick, and Jeanette Wyneken
Biology of Sharks and Their Relatives
Jeffrey C. Carrier, John A. Musick,
and Michael R. Heithaus
Early Stages of Atlantic Fishes: An Identification Guide
for the Western Central North Atlantic
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The Physiology of Fishes, Third Edition
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Biology of the Southern Ocean, Second Edition
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Biology of the Three-Spined Stickleback
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Huntingford
Biology and Management of the World Tarpon
and Bonefish Fisheries
Jerald S. Ault
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Methods in
Reproductive
Aquaculture
Marine and Freshwater Species
Edited by
Elsa Cabrita
Vanesa Robles
Paz Herráez
and
Boca Raton London New York
CRC Press is an imprint of the
Taylor & Francis Group, an informa business
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Library of Congress Cataloging-in-Publication Data
Methods in reproductive aquaculture : marine and freshwater species / editors,
Elsa Cabrita, Vanesa Robles, Paz Herráez.
p. cm. -- (Marine biology)
Includes bibliographical references and index.
ISBN 978-0-8493-8053-2 (hardback : alk. paper)
1. Fishes--Artificial spawning. 2. Fishes--Germplasm
resources--Cryopreservation. 3. Shellfish--Artificial spawning. 4.
Shellfish--Germplasm resources--Cryopreservation. I. Cabrita, Elsa. II. Robles,
Vanesa. III. Herráez, Paz. IV. Title. V. Series.
SH155.6.M48 2008
639.3--dc22 2008013533
Visit the Taylor & Francis Web site at
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THE EDITORS
The editors have been working together in the field of reproductive aquaculture
for the last ten years. Numerous scientific publications have arisen as a direct result of
their intensive work on sperm and egg quality evaluation and in developing sperm
cryopreservation protocols for different fish species. The evaluation of cryodamage
and the improvement of fish embryo cryoresistance have also been a priority in their
research. Nowadays, Dr. Elsa Cabrita is a researcher associate in the Spanish National
Research Council ICMAN-CSIC, Spain, where she works on fish reproductive
physiology. Dr. Vanesa Robles is a researcher associate in the Centre of Regenerative
Medicine in Barcelona CMR(B), Spain, where she works in fish germ cells and stem cell
biology. Both have worked previously at the University of León, Spain, and then
moved to the Center for Marine Sciences in Faro, Portugal, where they have continued
to develop research in the cryopreservation area. Dr. Paz Herráez is a professor in the
Department of Molecular Biology at the University of León, Spain, where she teaches
biology of reproduction and cellular biology. She is leader of the Cryobiology Group
and she has been responsible for the training of several doctorate and master’s degree
students.
METHODS IN REPRODUCTIVE AQUACULTURE:
MARINE AND FRESHWATER SPECIES
Gamete quality and management
PREFACE
The idea of writing this book came to us some years ago when we realized that we
had the conditions and opportunity to be involved in such a project. I remember
arriving at the lab and discussing this with my colleagues and we were all very
enthusiastic about the idea of publishing a book on gamete quality and
cryopreservation, our initial idea and objective at that time. The following months
were of hard work, especially after the publisher encouraged us to write a wider
overview of reproductive aquaculture. Actually, I must confess that at the time we
were not aware of what that decision would imply, and today, after looking through
these pages, we can say, with great satisfaction, that it was all worth the effort. The
fact that over 80 authors contributed to this unique project encouraged us to carry on
and overcome the difficulties we encountered. Some of you know the problems we
have had to face during these long months, especially in coordinating all the work
long distance.
Methods in Reproductive Aquaculture was written by several authors in response
to all the questions raised concerning gamete quality and management. Reproduction
has been a focus of research and interest in several marine and freshwater species.
Many species have been reproduced in captivity, most of them with interest in
aquaculture, fisheries or conservation. While the majority of these techniques have
been applied with some success under field conditions, some aspects related to
broodstock reproduction have been unattended. Gamete quality and management are
two important aspects in fish reproduction that deserve special attention, particularly
if we consider that both are requirements for the production of new individuals. This
constitutes the main focus of the book and all our efforts were directed towards
providing consistent and sound information, new objectives and areas of application
and research, and principally new tools to deal with some aspects in reproduction of
aquatic species.
Not all sectors have access to the considerable amount of information available
on fish reproduction, which is also sometimes difficult to process. Although several
books containing a wide range of subjects related to reproduction have been published,
we have attempted to focus on those aspects that we believe have been neglected.
This is the aim of our book, to reflect some of the aspects considered important in
the reproduction of marine and freshwater species, presenting all the available
information in an easily understandable way. To accomplish these objectives, the
book is divided into five sections. The first section is a review of the basic methods
and techniques for gamete extraction, spawning stimulation and stripping. The second
deals with sperm and egg quality and is focused on gamete characteristics and methods
used to evaluate quality; a third section includes techniques used for artificial fertilization
and the procedures for obtaining modified offspring (androgenesis, polyploids, etc.)
and the fourth section is a review of methods and advancements in gamete and
embryo preservation and storage. The last section includes several protocols for sperm
cryopreservation in different aquatic species. As our background is mostly in the area
of cryobiology, I must say that putting all this material from different authors together
and coordinating this section gave us particular pleasure.
The first part provides basic knowledge of gamete extraction methods, mainly
sperm collection and egg stripping in different species. Also, several techniques of
spawning stimulation (in males and females) by photoperiod and/or temperature control
and hormonal treatments are described. This section could be of special value for
teachers and students, clarifying concepts and specific techniques. For aquaculturists
and researchers it could be of great value in solving problems related to their broodstock
management and research.
The second section is based on sperm and egg characteristics commonly used
for quality assessment. Gamete quality is associated with larval quality and is a
requirement not only in production but also in several fields of research such as
embryology, genetics, biotechnology and cryopreservation, among others. Sperm cell
morphology, physiology and metabolism have been a focus of research basically in
cryopreservation studies. In this section, sperm analysis will also be considered from
a practical point of view in the management of broodstock conditions or in the selection
of appropriate breeders. Egg quality is a requirement for fertilization success and
several biomarkers such as egg morphological characteristics, enzymatic and metabolic
markers and novel molecular markers are discussed.
The third section of the book provides information on artificial fertilization from
normal practice used in fertilization trials to more specific fertilization procedures
allowing the manipulation of the normal process to produce more interesting progeny
from the viewpoint of aquaculturists and researchers.
In the fourth section the different chapters try to present an overview of methods,
research activities and directives in the management of gametes using biotechnology
tools to store the breeders’ reproductive potential for longer or shorter periods of time.
An introduction to cryopreservation procedures both in sperm, oocytes and embryos
is given as well as to materials and facilities required for gamete preparation and
freezing-thawing procedures. During recent years, fish sperm cryopreservation has
become an important tool in aquaculture facilities and conservation programs. It can
be used by fish farmers to facilitate daily procedures and increase production as well
as preserving genetic heritage, particularly of those individuals presenting desirable
characteristics. From a conservation point of view it enables the preservation of genetic
material from endangered species. For this reason we decided to incorporate a special
section describing specific protocols for the cryopreservation of sperm from several
species. Sperm cryopreservation protocols are detailed for fifty-six species, several
marine, freshwater, anadromous and catadromous species of teleosts, chondrosts,
molluscs, decapods or equinoderms. Each protocol includes three sub-parts. A general
description of the relevance of the species and sperm particularities is made. A detailed
description of the freezing-thawing protocol is given, including equipment and reagents
used, sperm extraction method, sperm extender and cryoprotectants, dilution rates,
sperm characteristics such as cell concentration, plasma osmolality and pH, sperm
loading and freezing/thawing rates, and so on. This information will be very useful,
particularly for people working in different fields and who are only interested in using
sperm cryopreservation as a tool.
In conclusion, this book provides information on endangered, highly profitable
species for aquaculture or fisheries and species with high potential in laboratory
research, constituting a complete guideline in an important aspect of fish reproduction-
gamete quality and management. This is the first practical guide in reproduction of
aquatic species that includes cryopreservation as one of the techniques in gamete
management. It establishes the basis for the reproduction of aquatic species, making it
accessible to interested parts, from fish farmers to scientific researchers and from
teachers to students, therefore having a wide range of potential readers.
We hope you enjoy reading this book as much as we have compiling it.
Elsa Cabrita
Vanesa Robles
Paz Herráez
