ILAR Journal Online, Volume 41(4) 2000: Cryobiology of Embryos, Germs Cells, and Ovaries

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ILAR Journal V41(4) 2000
Cryobiology of Embryos, Germ Cells, and Ovaries

Introduction
Genome Resource Banking of Laboratory Animal Models
J.K. Critser and Robert J. Russell

J.K. Critser, Ph.D., is Director of the Cryobiology Institute and Professor in the Department of Pediatrics and Immunology and Microbiology, Indiana University Medical School, Indianapolis, Indiana. Robert J. Russell, D.V.M., is Director of Laboratory Animal Medicine at Harlan Sprague Dawley, Inc., Indianapolis, Indiana.

"There can be no doubt that above all we, as a society and unified scientific community, must first understand the crucial need for genetic resource conservation. Given that, then the natural question becomes where do we go from here? The answer is to first identify needs and then logical priorities and strategies followed by implementation. . . . There is a fundamental need to know what is out there, its character, its usefullness, and how to protect it" (Wildt 1997, pp. 429-431).

These concepts were stated in the broad context of identifying and preserving biodiversity. However, they apply equally well to the specific case of preserving genetic resources and related issues that are currently exploding in laboratory animal research. This concept of an "explosion" of increasing numbers of new genotypes of laboratory animals brings with it a sudden increase in the number of new phenotypes. Although our understanding of the genetic component of this phenomenon is relatively well characterized, the phenotype counterpart remains much less well understood. In this regard, the scientific community is creating a vast number of new animal models, but it lags far behind in comprehensively phenotyping. However, this seemingly abundant supply of resources has led us as a laboratory animal research community into a situation not unlike that facing those who are attempting to address the loss of natural biodiversity in that we do not have a fundamental understanding of ". . . what is out there, its character, its usefulness and [whether to] protect it."

In this regard, the laboratory animal research community can learn much from previous efforts by several gropus to develop the concept, policies, and infrastructures associated with genome resource banks (GRBs¹). The first GRBs were established for domestic animal genetic management programs following the development of successful methods to cryopreserve domestic bull spermatozoa by Chris Polge and colleagues (Polge and Lovelock 1952: Polge and Rowson 1952). Starting in the 1950s, breeding programs for dairy and beef cattle by artificial insemination (AI¹) exploited this technology. The the 1970s, more than 10 million dairy and 2 million beef cattle were bred by AI with frozen-thawed semen (Gunasena and Critser 1997; Herman 1988). During the early 1970s, mammalian embryo cryopreservation was established in the laboratory of Peter Mazur at Oak Ridge National Laboratory (Oak Ridge, Tennessee) for mouse embryos (Whittingham et al. 1972). One year later, the first calf born from a cryopreserved embryo was reported (Wilmut and Rowson 1973). Since then, live births from many species using frozen-thawed embryos have been reported (Leibo 1986). In addition to processing and storing spermatozoa and embryos, GRBs often store additional cells and tissues such as ovarian tissue, blood cells, and skin biopsies, which can be used for rederivation, infectious pathogen screening, and somatic cell nuclear transfer, respectively. Currently the United States Department of Agriculture, under a Congressional mandate in 1990 (PL 101-624), has established a National Animal Germplasm program to identify and preserve potential important genetic resources from all of the major domestic animal agriculture species.²

In addition to the historical efforts that have been responsible for establishing GRBs for domestic animal agriculture, conservation biologists have developed similar strategies to preserve genetic resources from rare and endangered populations (Wildt 1992, 1997, 2000; Wildt and Seal 1994; Wildt et al. 1997). In this regard, the conservation biology community has long sought to develop multiple levels of programs to protect and preserve the Earth's biodiversity. In general, two categories of approaches have been applied in this context: (1) in situ programs, which often involve preservation or restoration of large areas of protected habitat; and (2) ex situ programs, which most commonly involve zoos, botanical gardens, aquaria, or private farms for the preservation of populations outside their natural habitat. More recently, the development of GBRs for ex situ preservation of biodiversity has been evolving, provoked by international discussions under the umbrella of the Conservation Breeding Specialist Group of the International Union for the Conservation of Nature and Natural Resources' Species Survival Commission. Through international efforts, Conservation Breeding Specialist Group stakeholders have made significant progress in the last decade toward identifying key concepts related to the successful establishment and operation of this approach to preserving important genetic resources. A synopsis of these concepts is presented in Table 1 (see also Wildt 2000). To date there are hundreds of species for which some form of cryopreserved biomaterial has been entered into a conservation-oriented GRB (D.E. Wildt, Smithsonian Institution, Fort Royal, Virgnia, personal communication, 1999).

Although not usually described in terms of GRBs, another area in which cryopreservation of reproductive cells and tissues are commonly used is the area of human reproductive medicine. As with domestic animal agriculture and conservation biology efforts to develop a "systematic and organized collection, storage, and use of biological materials," human assisted reproductive technology (ART¹) laboratories routinely process and store human spermatozoa and embryos as part of the overall approach to the treatment of infertility. The successful methods developed to cryopreserve domestic animal spermatozoa were subsequently applied to human spermatozoa and the creation of human sperm banks. A recent review of human sperm banking in the United States cited as many as 450 operations distributing up to 100,000 anonymous donor samples per year for use with one or more ARTs, such as artificial insemination or in vitro fertilization (Critser 1998). In addition, human ART laboratories routinely perform crypreservation of supernumerary embryos. There has been a marked increased in the number of children born from this procedure: approximately 300 in 1990, approximately 800 in 1993, and more than 2000 in 1997, the last year for hich these data are available (CDC 199; Gunasena and Critser 1997). As with the domestic and wildlife species GRBs, the human clinical ART laboratories have developed efficient procedures for the procurement, processing, storage, and distribution of human reproductive cells and tissues as an integral part of the overall management of patients and genetic resources.

Maintenance of stocks, strains, and lines of laboratory animals inherently share many critical aspects with GRBs established for other fields of science and medicine. As with the GRBs established for the maintenance of domestic animal germplasm and those for conservation biology, there is the initial, critical aspect of selecting which species and populations to sample and preserve. Unfortunately, infrastructural resource limitations necessitate selection of some populatoins and the de facto exclusion of others. Which populations of swine or cattle to preserve, or which endangered species to dedicate time and effort to preserving are often difficult decisions that require utmost care and access to information. However, key informatoin and technology are often lacking, making the process all the more difficult. In the context of laboratory animal research GRBs, processes for selecting specific stocks, strains, and lines of animals to be maintained and preserved must be predicated on meeting the research needs of the biomedical research community generally as well as individual investigators specificaly. Development of these selection processes requires evaluation of many factors, including the usefulness of the model, availability of both animals and effective methods for ART, and cryopreservation of reproductive cells and tissues.

Scope of Subsequent Journal Articles

The articles in this issue provide an overview of the current science and technology for the cryopreservation of murine reproductive cells and tissues. The information highlights the areas currently available for genome resource banking (e.g., mouse embryos) andother areas that require significantly greater research efforts before their application will become routine (e.g., rat spermatozoa). Gao and Critser (2000) discuss the basic science behind cryopreservation and provide the reader with an understanding of the complexities inherent in designing optimal methods for different cell types from various species. This information provides insights into the often-encountered perplexing problem in which cryopreservation methods for a particular species' cell type may work well, whereas the same procedure applied to a related species' cell type may simply not work. Critser and Mobraaten (2000) present information regarding the current status of the cryobiology and cryopreservation of mouse and rat spermatozoa. The authors provide an introduction to the importance of fundamental cryobiology and its usefulness in developing optimal methods of cryopreservation. They also discuss current approaches to and efficiencies of freezing mouse sperm while indicating that no successful methods are currently available for rat spermatozoa. Agca (2000) discusses two cutting-edge issues that involve the use of ovarian tissue and oocyte cryopreservation for preservation of genetic resources. In this article, the recent advances regarding ovarian tissue cryopreservation and transplantation are contrasted with the issues surrounding the continuing difficulties with oocyte cryopreservation. The article by Rall and colleagues (2000) describes the mouse and rat embryo cryopreservation program established at the National Institutes of Health. Information from this program provides critical insights into current efficiency problems and general methods associated with this most commonly used approach for murine genome resource banking. Wildt (2000) presents a system for genome resource banking in the context of wildlife research and conservation. Here the intricacis of developing and comprehensive and integrated "bank" or repository for genetic resources are discussed. Such banking provides an excellent model system for developing counterpart GRBs for the laboratory animal community.

References

Agca Y. 2000. Cryopreservation of murine oocyte and ovarian tissue. ILAR J 41:207-220.

CDC [Centers for Disease Control and Prevention]. 1999. 1997 Assisted Reproductive Technology Success Rates: National Summary and Fertility Clinic Reports. Washington DC: GPO.

Critser JK. 1998. Current status of semen banking in th USA. Hum Reprod 13(Suppl 2):55-65.

Critser JK, Mobraaten LE. 2000. Cryopreservation of murine spermatozoa. ILAR J 41: 197-206.

Gao D, Critser JK. 2000. Mechanisms of cryoinjury in living cells. ILAR J 41:187-196.

Gunasena KT, Critser JK. 1997. Utility of viable tissues ex vivo: Banking of reproductive cells and tissues. In: Karow AM, Critser JK, eds. Reproductive Tissue Banking: Scientific Principles. San Diego: Academic Press. p 2-17.

Herman HA. 1988. Improving Cattle by the Millions: NAAB and the Development and Worldwide Application of Artificial Insemination. 2nd ed. Columbia MO: University of Missouri Press. p 36-49.

Leibo SP. 1986. Cryobiology: Preservation of mammalian embryos. Basic Life Sci 37:251-272.

PL [Public Law] 101-624. November 28, 1990. 104 STAT.3744, Title XVI, Subtitle C: National Genetic Resources Program. Appendix I: Establishment, Purpose, and Functions of the National Genetic Resources Program. 7USC5841.SEC 1632.

Polge C, Lovelock JE. 1952. Preservation of bull semen at -70ºC. Vet Rec 64:396-397.

Polge C, Rowson LEA. 1952. Fertilizing capacity of bull spermatozoa after freezing at -79ºC. Nature 169:626-627.

Rall WF, Schmidt PKM, Lin X, Brown SS, Ward AC, Hansen CT. 2000. Factors affecting the efficiency of cryopreservation and rederivation of rat and mouse models. ILAR J 41:207-213.

Whittingham DG, Leibo SP, Mazur P. 1972. Survival of mouse embryos frozen to -196ºC and -269ºC. Science 178:411-414.

Wildt DE. 1992. Genetic resource banking for conserving wildlife species: Justification, examples and becoming organized on a global basis. Anim Reprod Sci 28:247-257.

Wildt DE. 1997. Genome resources banking: Impact on biotic conservation and society. In: Karow AM, Critser JK, eds. Reproductive Tissue Banking: Scientific Principles. San Diego: Academic Press. p 399-439.

Wildt DE. 2000. Genome resource banking for wildlife research, management, and conservation. ILAR J 41:228-234.

Wildt DE, Seal US. 1994. Population Biology Aspects of Genome Resource Banking. Apple Valley MN: IUCN/SSC Conservation Breeding Specialist Group.

Wildt DE, Rall WF, Critser JK, Monfort SL, Seal US. 1997. Genome resource banks: "Living collections" for biodiversity conservatoin. BioScience 47:689-698.

Wilmut I, Rowson LEA. 1973. Experiments on the low termperature prn of cow embryos. Vet Rec 92:686-690.

¹ Abbreviations used in this introduction: AI, artificial insemination; ART, assisted reproductive technologies; GRB, genome resource bank.
2For more information, contact the National Genetic Resources Advisory Council, Room 323-A Whitten Federal Bldg., 1400 Independence Ave., SW, Washington, DC 20250-0300. Tel.: 202-205-7835; Fax: 202-690-1434;<http://www.ars.grin.gov/ngrac/farmbill.htm>; or the USDA National Animal Germplasm Program: <http://www.ars-grin.gov/nag/>

Table 1 Factors to be considered in developing a genome resource bank (GRB) for laboratory animal models^a

Summary	Briefly describe the justification, goals, and overall conservation plan in the context of a GRB.
Justification	Describe the usefulness of the model to biomedical research.
Current knowledge of life history and reproduction	Assemble informaiton on sexual maturity and reproductive performance (e.g., litter size and embryonic and perinatal mortality). Indicate reproductive success as influenced by genetic, nutritional, disease, and management events. Describe extent of technology available for monitoring/managing animal health.
Current knowledge of assisted reproduction	Indicate prior success with cryopreservation of spermatozoa and embryos. Indicate prior success with in vitro fertilization and embryo transfer.
Status of the model	Indicate known or predicted animal numbers in various laboratories or repositories.
Accessibility of existing animals for banking	Identify accessibility of populations and individuals including ages and numbers of males and females.
Type and amount of germplasm to be preserved	Define short- and long-term management goals. Describe how the banking program will meet stated management and genetic goals. Calculate the minimum number of available individuals (founders or founder lines) to meet planned objectives. Identify the biomaterials to be preserved and stored (i.e., spermatozoa, ovarian tissue, embryos, tissues). Using computer modeling, calculate the amount of material needed from available individual founders, over a specific interval, to meet planned objectives.
Technical germplasm collection, processing,and storage	Indicate the optimal technology to be used based on previous research efforts. Describe how the health status of donors is to be determined to prevent disease transmission via movement of germplasm or othermaterials. Assemble the information necessary to identify precisely all stored aliquots of each biological material stored in a central database. Establish a labeling procedure containing information that is placed on each stored sample container. Identify primary and secondary locations for stored materials and the database. Describe the quality control program to be used.
Use and distribution	Indicate the circumstances under which stored materials will be provided and whether there will be a charge for distribution. Indicte the strategy for tracking follow-up information regarding the use of the distributed materials.
Ownership	Determine ownership of stored materials. Determine ownership of resulting offspring. Define how patents resulting from research using this material will be handled.
Resourcs and funding	Define personnel resources and expertise available for each phase of the banking process. Define required facilities resources (buildings, equipment, supplies). Identify funding for long-term storage and distribution. Develop a plan to ensure the transferability of the stored collection if those responsible are unble to maintain the bank in perpetuity.

^aModified from Wildt DE. 1997. Genome resource banking: Impact on biotic conservation and society. In: Karow AM, Critser JK, eds. Reproductive Tissue Banking: Scientific Principles. San Diego: Academic Press. p 399-49.