Advances in World Aquaculture, Volume 7

Cryopreservation in Aquatic Species 2nd Edition

A comprehensive overview of current practices, programmatic development and future directions for cryopreservation of gametes, embryos and larvae of aquatic species

Edited by

Terrence R. Tiersch and Christopher C. Green Published by

Advances in World Aquaculture, Volume 7

Cryopreservation in Aquatic Species, 2nd Edition Edited by Terrence R. Tiersch and Christopher C. Green Aquaculture Research Station Louisiana Agricultural Experiment Station Louisiana State University Agricultural Center Baton Rouge, Louisiana 70803

Published By The World Aquaculture Society

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Dr. Craig L. Browdy Managing Book Editor The World Aquaculture Society Copyright

2011 by

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Table of Contents Preface to the Second Edition .................................................................................................... xxiii Terrence R. Tiersch Acknowledgments for the Second Edition ................................................................................. xxvii Terrence R. Tiersch and Christopher C. Green Acknowledgments for the First Edition.................................................................................... xxviii Terrence R. Tiersch and Patricia M. Mazik Foreword to the First Edition .................................................................................................... xxix William E. Knapp Introduction......................................................................................................................................1 Terrence R. Tiersch

I. GONADS AND GAMETES ....................................................................................................18 Gonads and Gametes of Fishes......................................................................................................19 Harry Grier and Carole Neidig Spermatogenesis in Fish: A Structural and Functional Basis for Applied Research ....................33 Marcelo C. Leal, Rafael H. Nóbrega and Luiz R. França Induced Ovulation and Spermiation, and Factors Influencing Gamete Quality of Fishes ...........37 Edward Donaldson, Igor I. Solar and Brian Harvey Biochemical Characteristics of Seminal Plasma and Spermatozoa of Freshwater Fishes ...........46 Andrzej Ciereszko, Jan Glogowski and Konrad Dabrowski Sperm Nuclear Basic Proteins and Sperm Chromatin Organization in Fish ................................80 Juan Ausió, Núria Saperas and Manel Chiva Spermatozoal Ultrastructure of Ark Clams of the Superfamily Arcacea .......................................92 Wan-Xi Yang and Jun-Quan Zhu Physiological Modifications to be Considered for Amphibian Sperm Cryopreservation ...........100 Darío Krapf, Emma D. O'Brien and Silvia E Arranz Evaluation of Extenders for Refrigerated Storage of Koi Carp and Goldfish Sperm .................107 Donald W. Glenn III, R. Paul Lang and Terrence R. Tiersch

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Channel Catfish Pituitary as a Spawning Aid .............................................................................125 Christopher C. Green and D. Roger Yant Ultrasonographic Monitoring of Channel Catfish Ovarian Development ..................................134 Noel Novelo, Douglas Kuenz, Christopher C. Green and Terrence R. Tiersch

II. GAMETE QUALITY ...........................................................................................................145 Sperm Quality Evaluation for Broodstock Improvement .............................................................146 Elsa Cabrita, Vanesa Robles, Carmen Sarasquete and Paz Herráez Estimation of Fish Sperm Concentration by Use of Spectrophotometry .....................................162 Rafael Cuevas-Uribe and Terrence R. Tiersch Flow Cytometry for the Assessment of Sperm Quality in Aquatic Species ..................................201 Jonathan Daly and Terrence R. Tiersch Measurement of Milt Quality and Factors Affecting Viability of Fish Spermatotzoa .................208 Dale C. Honeyfield and William F. Krise Sperm Quality Assessment in Mammals by Flow Cytometry.......................................................219 Phillip Purdy Male Germplasm in Relation to Environmental Conditions: Synoptic Focus on DNA ...............227 Jill A. Jenkins Application of Computer-assisted Sperm Analysis (CASA) to Aquatic Species ..........................240 Huiping Yang and Terrence R. Tiersch Computer-aided Semen Analysis .................................................................................................255 Diarmaid H. Douglas-Hamilton, Kate M. Craig and Tom G. Kenny Microfluidic Mixers for Standardization of Computer-Assisted Sperm Analysis ........................261 Daniel S. Park, Christian Quitadamo, Terrence R. Tiersch and W. Todd Monroe

III. BASICS OF CRYOPRESERVATION .............................................................................273 Principles of Embryo Cryopreservation ......................................................................................274 Richard S. Denniston, Sherri Michelet, Kenneth R. Bondioli and Robert A. Godke Cryopreservation of Cattle Sperm ...............................................................................................291 John E. Chandler and Robert A. Godke

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Sources of Variation in Cryopreservation ...................................................................................298 S. P. Leibo Non-Equilibrium Vitrification: an Introduction and Review of Studies Done in Fish ................309 Rafael Cuevas-Uribe and Terrence R. Tiersch The Use of Calorimetry for Subzero Cryobiological Measurements...........................................325 Ram V. Devireddy

IV. CRYOPRESERVATION OF SPERM OF VERTEBRATES .........................................336 Preliminary Investigations on Sperm Cryopreservation of a Stingray, the Sparsely Spotted Stingaree....................................................................................................337 Jonathan Daly, Michael K. Holland and David B. Galloway Amphibian Conservation and Cryopreservation of Sperm, Cells and Tissues ............................345 Robert Browne and Chester R. Figiel Jr. Cryopreservation of Sturgeon and Paddlefish Sperm .................................................................366 Steven D. Mims, Ludmila I. Tsvetkova, William R. Wayman, Ákos Horváth, Béla Urbányi and Boris Gomelsky Cryopreservation of Sperm of the Endangered Razorback Sucker .............................................381 Terrence R. Tiersch, Chester R. Figiel Jr., William R. Wayman, J. Holt Williamson, Gary J. Carmichael and Owen T. Gorman Current Status of Sperm Cryopreservation in Siluriform Catfishes ............................................387 Ana T. M. Viveiros Cryopreservation of Sperm of Asian Catfishes Including the Endangered Mekong Giant Catfish ..................................................................................................................398 Krit Mongkonpunya, Thusanee Pupipat and Terrence R. Tiersch Cryopreservation Protocols for Sperm of Salmonid Fishes ........................................................409 Franz Lahnsteiner Cryopreservation of Sperm of Steelhead Rainbow Trout after Refrigerated Storage .................421 Joseph G. Cloud Cryopreservation of Sperm of the Endangered Apache Trout.....................................................425 Robert E. David, Lawrence J. Wirtanen and Michael A. Ternes Cryopreservation of Sperm of Striped Bass and White Bass .......................................................430 George G. Brown and Lynda D. Brown

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Sperm Cryopreservation in Biomedical Research Fish Models ..................................................439 Huiping Yang and Terrence R. Tiersch Cryopreservation of Striped Bass Spermatozoa ..........................................................................455 L. Curry Woods III Cryopreservation of Sperm of Some Marine Fishes ....................................................................459 Jin-Chywan Gwo

V. CRYOPRESERVATION OF EGGS AND EMBRYOS OF FISHES ..............................482 Problems and Prospects in Cryopreservation of Fish Embryos ..................................................483 Mary Hagedorn and Fritz W. Kleinhans New Approaches in Cryopreservation of Fish Embryos..............................................................503 Amrit Bart Cryopreservation of Sperm and Eggs of African Catfish Formerly Known as Sharptooth Catfish .......................................................................................................................513 Béla Urbányi, István Magyary, Ákos Horváth, B. Baranyai and András Dinnyés Cryopreservation of Gametes and Embryos of Cyprinid Fishes .................................................525 István Magyary, Béla Urbányi, Ákos Horváth and András Dinnyés Cryopreservation of Eggs and Embryos from Aquatic Organisms .............................................539 Jin-Chywan Gwo

VI. CRYOPRESERVATION OF GAMETES AND EARLY LIFE STAGES OF INVERTEBRATES ...................................................................................................................561 Cryopreservation of Molluscan Sperm: Pacific Oyster, Green-lipped Mussel, and Paua Abalone ........................................................................................................................562 Serean L. Adams, John F. Smith, H. Robin Tervit, Lindsay T. McGowan, Rodney D. Roberts, Achim R. Janke, Nick G. King, Samantha L. Gale and Stephen C. Webb Cryopreservation of Macha Surf Clam Spermatozoa ..................................................................574 Enrique Dupré and Alicia Guerrero The Need for Standardization in Cryopreservation: a Case Study with Oysters ........................581 Qiaoxiang Dong, Changjiang Huang and Terrence R. Tiersch Cryopreservation of Sperm and Larvae of the Eastern Oyster....................................................594 Carmen G. Paniagua-Chavez, John T. Buchanan, John E. Supan and Terrence R. Tiersch

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Cryopreservation of Eggs and Embryos of Shellfish ...................................................................604 Ta-Te Lin and Nai-Hsien Chao Cryopreservation of Pacific Oyster Oocytes ...............................................................................616 Serean L. Adams, H. Robin Tervit, Liliana Salinas-Flores, John F. Smith, Lindsay T. McGowan, Rodney D. Roberts, Achim R. Janke, Nick G. King, Stephen C. Webb and Samantha L. Gale Conserving Coral Reefs ...............................................................................................................624 Mary Hagedorn Cryopreservation of Juveniles of a Marine Polychaete ...............................................................631 Peter J. W. Olive and Wen B. Wang

VII. SOME BASIC METHODS ...............................................................................................645 Process Pathways for Cryopreservation Research, Application and Commercialization ..........646 Terrence R. Tiersch Research Methods for Cryopreservation of Sperm ......................................................................672 William R. Wayman and Terrence R. Tiersch Minimizing Microbial Contamination of Sperm Samples ............................................................684 Jill A. Jenkins Estimating Milt Quality of Salmonid Fishes ................................................................................688 Joseph G. Cloud Shipping of Refrigerated Samples ................................................................................................690 Terrence R. Tiersch Simple Extenders for Refrigerated Storage and Cryopreservation of Channel Catfish Sperm ................................................................................................................692 William R. Wayman, Chester R. Figiel Jr. and Terrence R. Tiersch Extender Solutions for Sperm of Salmonid Fishes .......................................................................698 Joseph G. Cloud Packaging and Identification of Sperm Samples .........................................................................701 Joseph G. Cloud Cryopreservation Methods for Sperm of African Catfish Formerly Known as Sharptooth Catfish .......................................................................................................................703 Béla Urbányi, András Dinnyés and István Magyary

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Cryopreservation Methods for Sperm of the Common Carp .......................................................706 István Magyary, András Dinnyés and Béla Urbányi Cryopreservation of Sperm of the Mekong Giant Catfish ...........................................................709 Krit Mongkonpunya, Thusanee Pupipat and Terrence R. Tiersch Cryopreservation Methods for Embryos and Larvae of the Pacific Oyster ................................712 Jin-Chywan Gwo Cryopreservation Methods for Embryos of the Pacific Oyster ....................................................714 Ta-Te Lin and Nai-Hsien Chao Fertilization Evaluation of Cryopreserved Sperm of the Atlantic Croaker .................................716 Jin-Chywan Gwo Production of Cyprinid Embryos for Cryopreservation Studies..................................................717 András Dinnyés, István Magyary and Béla Urbányi An Inexpensive Hatching Jar .......................................................................................................719 Donald W. Glenn III An Inexpensive Rearing Tank ......................................................................................................721 Mark C. Bates Performance of An Improved Temperature-Controlled Incubator ..............................................723 Donald W. Glenn III and Terrence R. Tiersch

VIII. GENETICS AND CRYOPRESERVATION .................................................................728 Genetic Consequences of Pooling of Sperm Samples ..................................................................729 Andrzej Ciereszko, Jan Glogowski and Konrad Dabrowski Utilization of Androgenesis for Strain Recovery from Cryopreserved Sperm .............................732 Gary H. Thorgaard, Paul A. Wheeler and Robert D. Fields Isolation of DNA from Cryopreserved Fish Sperm......................................................................738 Lynn Pittman-Cooley and Terrence R. Tiersch Preparation of Chromosomes from Cryopreserved Oyster Larvae .............................................743 Quiyang Zhang, Paul R. Lang and Terrence R. Tiersch Intracytoplasmic Sperm Injection (ICSI) in Fish .........................................................................748 Germán A. Poleo

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IX. PROGRAMMATIC DEVELOPMENT OF GERMPLASM REPOSITORIES ...........754 Ecological and Genetic Considerations for Collection of Gametes from Wild Fishes ...............755 Owen T. Gorman Development of Databases for Germplasm Repositories ............................................................760 Harold L. Kincaid The Role of Cryopreservation in Integrating Genetic and Genomic Research Programs at USDA-ARS and the Advancement of Domestication in Aquaculture ......................................770 Jeffrey Silverstein The USDA National Animal Germplasm Program and the Aquatic Species Collection.............774 Harvey D. Blackburn Germplasm Repository Programs in the United States Fish and Wildlife Service......................780 William R. Wayman The Application of Cryopreservation in Fish Genetic Conservation in North and South America .......................................................................................................................783 Brian Harvey The Northwest Salmonid Germplasm Repository ........................................................................789 Joseph G. Cloud, Robyn Armstrong, Paul A. Wheeler, Paul A. Kucera and Gary H. Thorgaard Cryopreservation of Germplasm for Effective Management of Animal Genetic Resources .......794 William Rall, Franziska Grieder and Michael C. Chang Xiphophorus Fishes: Varieties and Genetic Resources...............................................................796 Ronald B. Walter Sperm Cryopreservation and Storage at the Zebrafish International Resource Center .............809 Zoltan M. Varga and Monte Westerfield Medaka Cryopreservation and the Aquatic Biotechnology and Environmental Laboratory ......820 Richard N. Winn Commercial Application of Fish Sperm Cryopreservation for Management of Genetic Resources ........................................................................................................................828 John T. Buchanan and Valérie Barbosa-Solomieu

X. INTERNATIONAL PERSPECTIVES ...............................................................................834 Cryopreservation of Fish Sperm and Embryos in China .............................................................835 Song-Lin Chen, Xiang-Shan Ji and Yong-Sheng Tian

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Development of a Sperm Cryopreservation Approach to the Fish Biodiversity Crisis in Bangladesh...................................................................................................................................852 Mostafa A. R. Hossain, Md. Nahiduzzaman and Terrence R. Tiersch Status of Fish Germplasm and Cryopreservation in Africa ........................................................862 Kouassi S. Da Costa The Status of Cryopreservation of Aquatic Species in Mexico ....................................................870 Carmen G. Paniagua-Chávez, Gorgonio Ruiz-Campos and Marisela Aguilar-Juárez Current Status of Sperm Cryopreservation of Brazilian Characiform Fishes ............................875 H. P. Godinho and Ana T. M. Viveiros

XI. PERSPECTIVES FOR THE FUTURE APPLICATION OF CRYOPRESERVATION ..........................................................................................................885 Seminal Studies in the Cryopreservation of Fish Sperm .............................................................886 John H. S. Blaxter Lessons from the Cryopreservation of Livestock Sperm ..............................................................890 Lawrence A. Johnson Economics and Marketing of Cryopreserved Fish Sperm ...........................................................896 Rex H. Caffey and Terrence R. Tiersch Willingness-to-Pay for Specific Genetic Improvements for Aquaculture Species .......................917 Brian P. Boever, R. Wes Harrison and Terrence R. Tiersch Risk Considerations in the Application of Cryopreservation Techniques for Aquatic Species ............................................................................................................................931 Vincent A. Mudrak and Gregory L. Looney Infectious Disease and Quality Assurance Considerations for Transfer of Cryopreserved Fish Gametes .......................................................................................................939 Jill A. Jenkins Regulatory Considerations for Global Transfer of Cryopreserved Fish Gametes ......................960 Jill A. Jenkins The Relationship Between Conservation Policy and Aquatic Genetic Resources .......................977 Jennifer Long and Harvey D. Blackburn Ethical Considerations for the Application of Cryopreservation to Aquatic Species .................984 Stephen S. Wachtel and Terrence R. Tiersch

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Development of High-throughput Cryopreservation for Aquatic Species ...................................995 E Hu and Terrence R. Tiersch

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New Chapter

The Status of Cryopreservation of Aquatic Species in Mexico Carmen G. Paniagua-Chávez, Gorgonio Ruiz-Campos and Marisela Aguilar-Juárez

The Need for Cryopreservation of Aquatic Species in Mexico According to the National System of Information on Biodiversity (SNIB), Mexico has the second highest diversity of ecosystems in the world and is fourth in major species richness. Mexico is home to ~500 species important to fisheries, 600 species of plants that are used for reforestation, and some 4,000 species of plants known to contain medicinal properties. Hundreds of exotic species and other thousands with biotechnological potential are included in Mexican ecosystems (SNIB 2007). Despite recognition that Mexico is home to a large and unique biodiversity, the diversity of aquatic ecosystems is not acknowledged as well as terrestrial systems. Mexico is surrounded by four important seas (Pacific Ocean, Gulf of Mexico, the Caribbean, and Gulf of California). These regions contain species richness, endemism and diversity as high as those seen in terrestrial regions (Salazar-Vallejo and González 1993). Similar to other aquatic ecosystems around the world, the aquatic biodiversity of Mexico is threatened. Tourism development and constant demand for food have placed tremendous pressure on the Mexican coasts and seas (CONABIO-CONANP-TNC-PRONATURA 2007). The principal strategies developed by the Mexican government to conserve aquatic diversity were to assign protected areas and develop protection programs (CONABIO 2006). To date, only 1.4% of the marine environments are safeguarded under the protected area decree (CONABIO 2006). The official records for Mexico indicate that ~1,559 species of animals are registered under the categories of extinct, endangered, threatened or protected (Diario Oficial de la Federación 2007). From these records, 422 species are aquatic species, and 45% are fishes (Table 1). Table 1. Imperiled aquatic species from Mexico based on official records (Norma Oficial Mexicana 059-Ecol-2001, Diario Oficial de la Federación, 2002).

Group Amphibians Fishes Mollusks Crustaceans Corals Echinoderms Total

Extinct 0 11 0 0 0 0 11

Endangered 6 70 9 5 0 0 90

Threatened 42 74 0 7 0 0 123

Protected 149 30 8 3 7 1 198

Total 197 185 17 15 7 1 422

Also, the red list of threatened species published by the International Union for Conservation of Nature and Natural Resources (IUCN) lists ~2,945 species of animals from Mexico in one of 9 categories of risk (IUCN 2007). Consequently, the need to conserve aquatic diversity in Mexico is critical. In addition to the strategies currently used by the government, development of Paniagua-Chavez, C. G., G. Ruiz-Campos, and M. Aguilar-Juarez. 2011. The Status of Cryopreservation of Aquatic Species in Mexico. In: Cryopreservation in Aquatic Species, 2nd Edition. T. R. Tiersch and C. C. Green, editors. World Aquaculture Society, Baton Rouge, Louisiana. Pp. 870-874.

The Status of Cryopreservation of Aquatic Species in Mexico

Paniagua-Chavez et al.

cryopreservation of gametes and embryos can assist conservation of genetic resources of aquatic species and support conservation programs to safeguard endangered and threatened species. The paucity of information found on aquatic population dynamics, in combination with the shortage of protected areas and the increasing pressure on aquatic ecosystems due to human activity, make aquatic organisms very vulnerable in Mexico. The status of freshwater fishes has been better documented than that of marine species. One of the native freshwater fishes of northwestern Mexico with a high potential for aquaculture and conservation are the trout species of the genus Oncorhynchus. These species are distributed in the headwater streams of the Sierra Madre Occidental (SMO) and Sierra San Pedro Mártir (SSPM), within an altitudinal interval from 560 to 2,640 m above sea level (Ruiz-Campos et al. 2003). This genus is represented in Mexico by at least ten native taxa, of which only two have been taxonomically described: the Mexican golden trout (Oncorhynchus chrysogaster) (Figure 1, next page), from the SMO in Chihuahua, and the Nelson’s rainbow trout (Oncorhynchus mykiss nelsoni), from SSPM in Baja California (Behnke 2002, Ruiz-Campos et al. 2003, Hendrickson et al. 2003, 2006). The conservation status of this complex of species is vulnerable due to their confined distribution and low abundance within populations (Hendrickson et al. 2006, Jelks et al. 2008). This complex of species represents a spectrum of evolutionary relationship with genetic diversity achieved through 0.5 million years of adaptation and speciation. The genetic pool provided by this myriad of species could be used for genetic improvement and enrichment of the stocks of cultured rainbow trout, and in programs of recovery of endangered or threatened species. One of the main problems that confront commercial culture of exotic rainbow trout (O. mykiss irideus) in Mexico and elsewhere in the world is the erosion of genetic diversity of broodstocks due to repeated interbreeding of a limited stock of breeding adults. The effects of the inbreeding are reflected in high mortality due to susceptibility to disease and infections by parasites, and a lessened tolerance to daily changes of temperature and dissolved oxygen. The genetic diversity and physiological traits of cultured rainbow trout could be gradually improved by the programmed addition of genetic material from the native trout populations originating from the wide geographic range of northwestern Mexico. Therefore, the establishment of a germplasm bank for native trout populations of northwestern Mexico, provides a window of opportunity with several lines of application in the short-term or long-term such as: (1) preservation of genetic pools of different native trout populations, (2) enrichment of genetic diversity of cultured rainbow trout stocks, (3) establishment of a source of genetic material for ex situ reproduction of imperiled species, and (4) support of programs focused on reproduction and stocking of trout for sustained sport fishing. Preservation of the genetic resources of these trout species can thus provide a model for many other fish species in Mexico and elsewhere.

The Germplasm Bank of Aquatic Species of Baja California In 2005, the Germplasm Bank of Aquatic Species (GBAS) was inaugurated in Baja California with the purpose of conserving genetic resources of aquatic species. This project was supported by the Secretary of Environment and Natural Resources (SEMARNAT) and the National Council of Science and Technology (CONACyT). The GBAS began as a pilot project to collect genetic resources from the Baja Peninsula, which is the second longest and most isolated peninsula in the world (Grismer 2000) and contains unique and threatened

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Figure 1. (Top) Mexican golden trout (Oncorhynchus chrysogaster, 138 mm standard length) from the Sierra Madre Occidental, Chihuahua. (Bottom) Nelson’s rainbow trout (Oncorhynchus mykiss nelsoni, 148 mm standard length) from the Sierra San Pedro Mártir, Baja California. Photographs by G. Ruiz-Campos.

biodiversity. In the past 5 yr, collections of germplasm from five important biological or commercial species have been performed (Table 2). Table 2. Cryopreserved material stored in the Germplasm Bank of Aquatic Species of Baja California.

Common name Totoaba Rainbow trout Pacific oyster Red abalone California halibut

Scientific name Totoaba macdonaldi Oncorhynchus mykiss nelsoni Crassostrea gigas Haliotis rufescens Paralichthys californicus

Status Endangered Special protection Commercial Commercial Commercial

Material Sperm Sperm Oocytes Sperm Sperm

Sample collection is subject to reproductive seasonality for each species and government permits are required for collection of samples from endangered or protected species. Totoaba and Nelson’s rainbow trout are two endemic species, for which frozen sperm was used to fertilize eggs in 2009. Abalones and flounders are important commercial species that will also benefit from the bank. Frozen sperm from these species will be used for breeding when fresh sperm is not available.

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In addition to collections, different experiments need to be performed to determine optimal conditions for freezing of samples. Such experiments are being performed for sperm of white shrimp, Penaeus vannamei, and the sea cucumber, Cucumber apostichopus, and for sperm and embryos of red abalone Haliotis rufescens. The research developed at the GBAS is the product of collaborative work among Mexican universities including the Universidad Autónoma de Baja California (UABC). The Universidad Autónoma Nacional de México (UNAM), The Universidad Autónoma Metropolitana (UAM) and The Centro de Investigaciones en Matemáticas (CIMAT). This research has thus far focused on optimization of freezing protocols, reduction of pathogen transfer, determination of gamete quality, and development of information technologies. The urgent need to protect the biodiversity of Baja California presents a long list of species that must be considered for cryopreservation. Many mollusks (including all species of endemic abalone, two species of geoduck, and important clams), marine fish (sardines and yellow tail, among others) and freshwater fishes (such as endemic trout) will be cryopreserved. Perspectives for the Future of Cryopreservation of Aquatic Species in Mexico In Mexico, previous conservation of genetic resources has been limited almost entirely to commercial plant species. These duties are carried out principally by the Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP), the Centro Nacional de Mejoramiento del Maíz y Trigo (CIMMYT) and the Centro Internacional de Mejoramiento de Maíz y Trigo of the Universidad Autónoma de Chapingo (UACH). Most of the techniques to conserve these germplasm are based on cold storage of seeds. Cryopreservation programs for livestock are carried out in the Facultad de Veterinaria, Universidad Autónoma de Mexico (UNAM). Most of the cryopreservation is performed for bull, sheep, and horse, however, a well-established germplasm bank for domestic species does not exist although some companies cryopreserve sperm and embryos at small scale for commercial purposes (S. Rincón, Ganadería Romar; E. Tavitaz, Hacienda La Escondida, personal communications). The large-scale conservation of genetic resources of aquatic species is a new concept and is performed in Mexico only by the GBAS. Recently, efforts have been made by the government, research institutes, universities, and industry, to develop the first National Germplasm Bank (NGB) and to create a National Seed Center, and a Forest Germplasm National Center. The GBAS has been considered to participate in these challenges. This will be a good opportunity to expand the work performed by the GBAS, to cryopreserve important aquatic species from the entire country and to participate actively in the developing conservation programs of Mexico. Acknowledgments This work was supported in part by funding from Secretary of Environment and Natural Resources (SEMARNAT) and National Council of Science and Technology (CONACyT), Project number SEMARNAT-2002-C01-0393. We thank the Secretary of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA) for providing collecting permits used for the Germplasm Bank of Aquatic Species of Baja California.

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