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4 New Technology and Its Impact on Conservation New technologies are creating greater possibilities for preserv- ing and using animal genetic resources. New molecular ge- netic technologies can provide valuable tools for characteriz- ing and using genetic stocks. Of particular importance will be im- proved capacities to search preserved germplasm for useful genes that can be incorporated into existing breeding stocks. Improved methods for diagnosing and controlling diseases will significantly reduce barriers to exchanging germplasm across borders and thereby enhance the value of preserved stocks for future use. This chapter describes new and emerging technologies to quan- tify genetic variation, to transfer genes from one animal to another, and to optimize reproduction. With further development, many of these technologies could provide powerful tools for preserving and using livestock genetic resources. The chapter ends with a brief dis- cussion of the impact of health status on the movement and use of genetic resources that is followed by a set of recommendations. METHODS TO QUANTIFY GENETIC VARIATION Biotechnological methods that enable scientists to characterize an animal's genes and the gene pools of breeding populations are becom- ing a reality. These methods will aid in making decisions about the uniqueness of germplasm for preservation. The study of the struc- ture and function of genes at the molecular level (molecular charac- terization) in a breeding population can help determine the similarity ~7

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78 / Livestock of the genetic material carried by two or more populations and the genetic variation within a population. The identification of individual genes, on the other hand, will allow the genetic makeup of an animal (genotype), as distinguished from its appearance (phenotype), to govern the process of choosing the parents of the next generation (artificial selection). If genes for production traits can be identified, for ex- ample, they can be selected for even if they are not expressed in an individual animal. Alternatively, if they can be linked to genes with known locations on chromosomes and clear-cut phenotypes (marker loci), selection can be done using these points of reference. This allows selection for a desired character even though the genetic basis for the desired phenotypes may not be known. Molecular Quantification of Genetic Diversity and Distances Several techniques are used to estimate genetic differences be- tween populations. Each has its own set of assumptions and applica- bility to a given problem. All attempt to quantify genetic distance between populations, which is a measure of the number of genetic differences between two populations or species. Molecular techniques for estimating differences have been reviewed by Chambers and Bayless (1983), Net (1987a), and Sharp (1987~. These methods include DNA (deoxyribonucleic acid) sequencing, DNA-DNA hybridization, pro- tein electrophoresis, immunologic methods, and restriction fragment length polymorphisms (RFLPs). DNA sequencing involves determining the sequence of base pairs in sections of the DNA. Although techniques for automating DNA sequencing are being developed and improved, the process is still too expensive and time consuming to be routinely applied. A reli- able estimate of sequence divergence between two populations would require sequencing of many genes for many animals within each popu- lation. This is not currently feasible in animals, but it could become the main method for determining genetic distance in the future. DNA-DNA hybridization requires the isolation of DNA from two related genomes. The DNAis treated with heat, causing the DNA double-stranded helix to disassociate and become two single-strand DNA molecules. When the single-strand DNA from two different sources is combined, it will re-form into double-stranded DNA upon cooling. The stability of the re-formed double-stranded DNA de- pends on the exactness of the match. The extent of nucleotide differ- ences between the two different strands can then be approximated upon reheating and measuring the temperature at which the double strand dissociates again into single strands; this provides an index of relatedness and so, conversely, of genetic distances.

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New Technology and Its Impact on Conservation / 79 :~: :: :~ :~.~ ~ : it- : :::- an- i, ~,~-~--~- :~ ~ an. - ~::~ ~ ~.~ ~ -- - :~- ~ ~ ~ ~: ~ : ~ - ~ ,, Nisi ~- - ~ ,~ ~-.~. ....- ..-.,..-- ~ (~ -, -~-~-.~- i--, ~.-~.~ .-- -I--. ha-.... W~- ~,,.- ~.~ ,,,,,.,. .,~,~,,,.,,.,~,,~,~., ,~,~,~.,.,,.,,Kj,,.,, ~,~,,,., I, ., v i, i., . ,,,,,. ~ .,, ~.~,~, ~it. ,., , i. Restriction fragment length polymorphisms from five individuals in a popu- lation of crossbred cattle are revealed using two different restriction enzymes (EcoRI and BamHI) and a probe for the eye lens protein, a crystallin (CRYA1~. Alleles that are polymorphic are labeled a or b with each fragment size ex- pressed in kilobases (kb). In this example, a single probe and two restriction enzymes help distinguish four sites of genetic difference among the five indi- viduals. Source: Thielman, I. L., L. S. Skow, I. F. Baker, and I. E. Womack. 1989. Restriction fragment length polymorphisms for growth hormone, pro- lactin, osteonectin, a crystallin, ~ crystallin, fibronectin and 21-steroid hy- droxylase in cattle. Anim. Genet. 20:257-266. Reprinted with permission, O1989 by Blackwell Scientific. O'Brien et al. (1985) applied this technique to estimate the genetic distance between species of higher primates and carnivores and com- pared it with more classical estimates derived from protein variation (isozyme) analysis. Among the methods for estimating molecular distance, DNA-DNA hybridization produces data that more closely agree with those derived from evolutionary studies of primates. Ge- netic distances between these primates as measured by isozyme analysis are similar to those observed between cattle breeds (Sharp, 1987~. Thus, DNA-DNA hybridization might be useful for quantifying rela- tionships among breeds of livestock. Electrophoretic analysis of protein variation has been the most

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80 / Livestock popular technique for estimating genetic distances. It has been widely used for many years to detect genetic variation in populations at the molecular level (Harris, 1966; Lewontin and Hubby, 1966~. The method is based on the principle that proteins of differing sizes and struc- tures will have differing net charges, and thus differing mobility, when placed on a gel and allowed to move in response to an electric field. The proteins detected by electrophoresis are various enzymes, and a small amount of blood or tissue is a sufficient sample for measurement. In typical outbreeding mammalian populations, two or more alle- les were found at 15 to 25 percent of the gene loci examined (that is, they were polymorphic). The average heterozygosity in individuals over all loci is between 3 and 10 percent (Baccus et al., 1983; Nevo, 1978; Selander and Kaufman, 1973~. To date, about 30 polymorphic loci have been identified that can be used to analyze the genetic structure of livestock species. The identification of genetic variation by electrophoretic analysis underestimates the genetic variation at the DNA level. Only a small fraction of the genome codes for proteins, and thus, a random sample of the DNA is not examined. Even in the coding regions, sequence changes in DNA do not always lead to amino acid changes; thus, they will not be reflected as electrophoretic changes. Even if changes do occur, only those changes that affect the net charge of the protein will be detected. Nevertheless, sufficient protein variation can be found to quantify genetic distances (Nei, 1987a), and electrophoresis has been successfully applied to the study of genetic distances be- tween breeds (Glodek, 1982; Gonzalez et al., 1987; Kidd et al., 1980~. The detection of allelic variation in blood group proteins by im- munological techniques (Render, 1958; Stormont, 1959) is comparable to protein electrophoresis in application and limitations. The num- ber of identified variant proteins, however, is less. For example, only about 12 blood proteins have been identified in cattle (Standing Committee on Cattle Blood Groups, 1985~. Detection of variation by other meth- ods, such as the use of molecular markers, appears to have greater potential (Baker and Manwell, 1991~. A relatively new method, RFLP analysis, can be used to charac- terize populations. Restriction endonucleases are enzymes that cut DNA at specific sequences, called restriction sites (Zabeau and Rob- erts, 1979~. When genomic DNA is cut with one of these enzymes (more than 100 are commercially available), a mixture of DNA frag- ments of varying sizes results; these are called restriction fragments. Electrophoresis is used to separate the restriction fragments accord- ing to size. The fragments are then transferred to a filter by blotting (Southern, 1975), which produces a replica of the gel on which DNA

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New Technology and Its Impact on Conservation / 81 DNA hybridization can occur. Specific DNA sequences, known as DNA probes that are labeled with a radioactive isotope of phospho- rus (32p), bind to complementary DNA fragments on the filter. Posi- tions of the probes, which correspond to specific restriction fragments, can be determined using autoradiography. If, in two DNA samples, fragments of differing size result from the use of the same restriction enzyme, they reflect a polymorphism or difference in the base-pair sequence in the restriction site (that is, the site at which the enzyme cut the DNA). Such a polymorphism in a restriction site is evidenced by bands at different positions on the filter, because electrophoretic mobility is a function of fragment size. Assuming that the DNA of domestic animals proves to be as poly- morphic as that of humans, a polymorphic site should occur in one of every 100 base pairs (Teffreys, 1979~. The RFLP technology and its application to animal breeding were reviewed by Soller and Beckmann (1983~. The authors summarized data from humans, mice, and cattle and estimated that 5 to 10 per- cent of enzyme-probe combinations can be expected to reveal an RFLP. Consequently the level of genetic variation revealed by RFLP analy- sis far exceeds that from protein electrophoresis. Because the tech- nique is relatively new, very few applications have yet been made to estimating genetic distance. Mathematical models have been devel- oped, however, for estimating genetic divergence from restriction site polymorphisms (Nei, 1987b; Nei and Tajima, 1981~. Wainscoat et al. (1986) have applied these models to studies of relationships among human populations. As procedures for evaluating genetic distance between popula- tions are refined, they will provide much-needed information on the distribution of genetic variation within domestic species (Theilmann et al., 1989~. This information will greatly assist in making conserva- tion decisions by allowing more quantitative evaluation of the prob- ability that an endangered population contains alleles that are unique to the species. Studies to characterize the genetic structure of global livestock breeds should thus receive high priority. Characterizing Mitochondrial DNA In cells, DNA is also found outside the nucleus in small ablate bodies, the mitochondria. Mitochondrial DNA (mtDNA) is consider- ably smaller than nuclear DNA and is easily isolated. Consequently, mtDNA has been well characterized in most animals and is widely used in evolutionary studies. Mitochondrial DNA genes are trans- mitted through the female parent and so reveal only the maternal

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82 / Livestock lineage. This limits their usefulness in fully defining breeding popu- lations of livestock. The mitochondrial genomes of several livestock species, including cattle, have been sequenced. Developing Genetic Maps Gene mapping assigns a locus to a specific chromosome or deter- mines the sequence of genes and their positions relative to one an- other. Knowledge of RFLPs has greatly aided gene mapping (Beckmann and Soiler, 1983~. A genetic map saturated with RFLP markers at frequent intervals would ensure that an association could be detected between a marker and any important gene. No agriculturally impor- tant gene would be beyond the reach of marker-assisted selection. Individuals with desirable genes could be selected on the basis of markers even in the absence of phenotypic expression of the gene or of knowledge of its molecular basis. Rams carrying the Booroola gene for prolificacy, for example, could be identified without prog- eny testing their daughters if the gene were mapped near a marker. RFLPs appear to be widespread in all species, although only a small number have been defined in livestock to date (Foreman and Womack, 1989; Fries et al., 1989~. Applications of RFLP-assisted mapping to human genetics are reviewed by Watkins (1988~. More than 4,500 loci have been cataloged for the human genome, and the number is rapidly increasing (Human Gene Mapping 10, 1989~. The power of this technique for genomic analysis of livestock has only recently been recognized. Recent literature suggests a trend to exploit these markers, at least in cattle, for which more than 30 have been de- scribed to date (Beckmann et al., 1986; Fries et al., 1989; Hallerman et al., 1988; LeQuarre et al., 1987; Theilmann et al., 1989~. An even more powerful class of RFLPs, called variable number of tandem repeats (VNTRs), has contributed much to expanding the hu- man gene map (Nakamura et al., 1987~. Searches for VNTRs in live- stock are only beginning. The advantage of these markers is in their extreme variability; they exist as multiple alleles with a high prob- ability of being heterozygous in any given animal. Single-locus NTNTR probes have not been described for livestock, but the extensive re- striction fragment variation associated with these probes has been described (Vassar" et al., 1987~. The VNTR probes may be particu- larly useful in establishing parentage because they will be more simi- lar in closely related animals. Linkage groups refer to loci (genes or markers) that are in close proximity on the same chromosome and, therefore, are generally in- herited together. Genetic mapping of a linkage group (linkage map

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New Technology and Its Impact on Conservation / 83 ping) requires studying inheritance of the markers or phenotypes of interest over several generations. Genetic mapping is easier when multigenerational pedigrees of large families are available. Several linkage studies have been performed in livestock based on the small battery of markers provided by enzyme and blood group research (Andersson, 1986; Hines et al., 1969; Larsen, 1977~. These studies can now be replicated using the much larger number of gene markers provided by RFLP analyses to detect many more linkage groups. Physical mapping, which determines the precise location of a gene on a particular chromosome, can be accomplished in livestock spe- cies with the same technologies used for humans (Fries et al., 1988~. Mammalian chromosomes are sufficiently preserved across species to allow the identification of homologous (similar) segments between such diverse species as cattle and humans (Womack, 1987; Womack and Moll, 1986~. Their identification permits extrapolation of map- ping data from one species to another. This capability may be par- ticularly important to animal geneticists in light of the wealth of data being obtained from human genome mapping. The current activity in animal gene mapping suggests that useful maps for most of the livestock species will be developed by the mid- 1990s. In 1987, 50 genes had been mapped in cattle, 31 in sheep, 38 in pigs, and 21 in horses (Lalley et al., 1987~. By 1989, the number of genes mapped in cattle had already doubled. An extensive linkage map of the domestic chicken was published in 1987 (Somes, 1987~. DNA LIBRARIES AND GENE TRANSFER METHODOLOGIES Gene transfer involves identifying and isolating a gene from one genome and transferring it into the genome of another individual. Transfers may be within the same species or from one species to another. To date, most work on mammals has been with mice; how- ever, application of gene transfer technologies in livestock species, with the objective of improving production traits, is receiving greater attention (First, 1991; Gibson and Smith, 1989; Pursel et al., 1989; Smith et al., 1987; Wagner, 1986~. A closely related technology is the formation of DNA libraries, which has important potential for pre- serving germplasm. Genomic DNA and DNA libraries from different genetic sources also represent valuable research resources. Establishing an Animal Genomic Library An animal genomic library is a collection of cloned fragments representative of individual genomes. In a genomic library, genes

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84 / Livestock from an animal, in the form of segments of DNA, are incorporated into the genome of a simple organism, such as a bacteriophage. As the "carrier" organism multiplies, it duplicates its genome as well as the added segments. The collection of cloned DNA segments that comprise the genomic library, however, has no catalog; additional screening is needed to determine which genes are present and where they are located within the library. Cloning Genes in a Library A genomic library can be established by isolating DNA from vir- tually any tissue (for example, organs, blood, semen). The DNA is cut into small segments with specific DNA-cleaving enzymes (restric- tion endonucleases). Random DNA segments are then inserted into the intact genome of a virus (such as bacteriophage lambda), which can be rapidly reproduced in its bacterial host, Escherichia coli. Many copies of the bacteriophage lambda are made within each bacterium to yield multiple cloned copies of the inserted DNA segment. DNA segments, containing one or more genes, are inserted into separate lambda genomes. These bacteriophages can then be propagated sepa- rately as clones derived from the single "transgenic" (because it now contains genetic material transferred from the source animal tissue) lambda phage. The complete genomic library consists of a suspension of millions of transgenic lambda phage organisms, each carrying a gene or genes from the source animal. This suspension may be cryopreserved in- definitely. It may also be multiplied to produce additional copies of the library. When an appropriate volume of the transgenic bacterio- phage suspension is placed on a plate containing a layer of E. coli, each individual transgenic organism will form a separate colony (plaque). In theory each colony contains a piece of the original animal DNA and can be isolated for further growth and testing. These colonies represent individual "cloned" segments of DNA. Screening an Animal Genomic Library Screening a genomic library is the process of discovering which colony contains a particular DNA segment. All phages grown from that colony will contain the gene of interest: The screening of a genomic library requires a means of identify- ing the appropriate colony. This is accomplished by using a radioac- tively labeled DNA probe. A messenger RNA or segment of a known gene can be used to construct a DNA probe that has a nucleotide base sequence complementary to all or part of the DNA sequence of

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New Technology and Its Impact on Conservation / 85 interest. When placed on the growth plate containing the entire li- brary, the probe will bind only to colonies containing the comple- mentary DNA segment. If the plate containing the library is then placed over X-ray film, a dark spot will develop under that colony, thereby identifying it. Once a colony has been identified and isolated, large quantities of the organisms can be grown to increase the number of copies of its particular DNA segment. The DNA sequences can then be separated from the virus DNA using the same endonuclease originally employed in constructing the library. To isolate a single gene it may be neces- sary to obtain DNA segments from more than one-colony because intact genes are rarely found in a single clone. Using this technique, individual genes can be located, sequenced, studied, and used for gene transfer. Transfer of a Cloned Gene into Another Animal Line A cloned gene from a genomic library may be incorporated into the genome of another animal. In mammals the cloned DNA seg- ment is microinjected into the pronucleus of the fertilized egg. En- zymes for maintenance and repair of the embryonic DNA are be- lieved to splice the added gene into a chromosome. It is thereby permanently incorporated into the genetic composition of what is now termed a transgenic animal. Procedures for introducing cloned genes into avian species have also been developed during the past several years (Brumbaugh, 1989; Freeman and Bumstead, 1987; Cannon et al., 1990~. The percentage of embryos that are transgenic (containing the extraneous DNA segment) following microinjection of the eggs is low, generally less than 2 percent in livestock species. Because the site of the insertion cannot be controlled, the DNA segment may be incorporated anywhere in the genome. This may impair the function of another gene within the genome. Control of the insertion site has been achieved for some cloned genes in the mouse (Capecchi, 1989~. Although successfully incorporated, some transgenic genes may not be expressed. The value of those that are expressed will depend on the site (or tissue), time, and intensity of expression. So far, no trans- genic livestock have proved useful commercially, and many show seri- ous deleterious effects of the transfer (Purser et al., 1989~. Thus, al- though the transfer of genes as DNA from genomic libraries has been shown to be possible across species, much research and development work is needed to make it an effective tool for using animal genes. Animal genomic libraries may also provide a means of evaluat- ing preserved animal germplasm. If the DNA coding for specific

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86 / Livestock gene products can be identified, the DNA of live or cryopreserved stored germplasm could then be screened for potentially useful vari- ants of the gene. Specific alleles, identified by their DNA sequence, could then be bred into current stocks for evaluation. DNA Libraries as Supplements to Conservation Programs DNA should be considered only as an adjunct, rather than pri- mary, means of germplasm conservation. Regeneration of a living organism is not yet possible from DNA stores. In certain situations, however, preservation of DNA stores may be a reasonable and useful supplement to other efforts. For example, a sufficient DNA sample can be obtained from blood or sperm cells collected when taking semen samples for cryopreservation. The tissue sample could be frozen without further processing or processed to yield purified DNA for storage. Although a DNA sample can be obtained from several billion sperm cells or from 50 to 100 milliliters of blood, continuing studies of any population may require considerably greater quantities of sample DNA. This additional DNA could be derived from genomic libraries and propagated. If stores for a preserved population, such as those described above for sperm, decline, a genomic library should be es- tablished to preclude loss of potentially useful DNA. Identifying Quantitative Traits Techniques such as RFLP and hybridization analysis may be used to characterize the genomes of individuals in a specific population and to compare them with reference genomes from other popula- tions. These molecular descriptors may be of importance in selecting specific samples from frozen stores. However, these techniques focus on individual genes while most production traits are affected by many genes (quantitative traits), each with individual effects that are not easily detected. Methods are being developed in animals for identi- fying and tagging the different segments of the genome containing genes of moderate or large effects on a quantitative trait (Soiler and Beckmann, 1983~. This approach has proved successful in plants (Tanksley et al., 1989~. REPRODUCTIVE TECHNOLOGIES Until recently, genetic change within populations was, in general, relatively slow. The application of new reproductive technologies

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New Technology and Its Impact on Conservation / 87 and the increased international exchange of germplasm can now en- able rapid and dramatic changes. For example, in many countries, national chicken populations have changed practically overnight from genetically heterogeneous backyard fowl to selected homogeneous stocks raised under intensive conditions. Cryopreservation of Semen and Embryos The discovery in 1949 that semen could be protected from the harmful effects of freezing and thawing was an important develop- ment in the area of animal breeding. Considerable progress has been made since then in establishing procedures for freezing and storing the semen and embryos of a wide variety of mammalian species, usually In liquid nitrogen at-196C. Critical elements of cryopreservation are the development of suitable media to protect cells when frozen and stored, and procedures to ensure that the rates of cooling and warming prevent the detrimental effects of intracellular freezing. A good understanding of the processes responsible for freezing injury has emerged. Cryopreservation now offers a cost-effective way to retain a sample of the genetic diversity of a population of animals in suspended animation for indefinite periods of time. Current technology permits the cryopreservation of semen for A technician in a Brazilian lab- oratory tests a catheter before attempting embryo collection from endangered horses in Brazil. Credit: Elizabeth Henson, Cots- wold Farm Park.

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88 / Livestock most livestock species. In cattle, methods for semen collection and freezing are well established, and artificial insemination is widely used, especially for dairy cattle. Cryopreserved buffalo semen is commercially available. For sheep, pigs, goats, horses, and poultry, cryopreservation methods are not yet fully satisfactory, and frozen semen is not widely used commercially. For special applications with these animals, however, cryopreservation of sperm is feasible. For REPRODUCTIVE AND MOLECULAR TECHNOLOGIES PRESENT CONCERNS AND OPPORTUNITIES FOR CONSERVATION Concern about conserving genetic diversity in livestock species is largely a response to technological innovations that have allowed wide- spread dissemination of genetic material from a limited number of highly selected breeds and individuals. Prior to the last half of the twentieth century, the breeding structure of most livestock species was nearly ideal for maintaining genetic diversity. Livestock populations were widely dispersed geographically and strongly subdivided along regional and national lines. Movement of individuals among neighboring herds was common, but the international and transcontinental movement of livestock was relatively rare, in part because of difficulties in moving large numbers of breeding animals over long distances. Fear of intro- ducing exotic diseases also limited the movement of germplasm, as did the frequent inability of imported animals to survive and reproduce in foreign environments. More recently, however, many of the problems of germplasm trans- fer have been solved, and the global movement of germplasm has greatly accelerated. The greatest impact has come from the use of artificial insemination and embryo transfer using frozen semen and embryos. The genetic makeup of national livestock populations can be encom- passed by a few highly selected animals. Internationally, these tech- nologies, coupled with improved diagnostic tests and embryo washing procedures to reduce risks of disease transmission, will soon allow individual animals to have a far-reaching global genetic influence. Tech- niques for in vitro oocyte maturation and fertilization and for embryo cloning promise to expand the potential impact of individual parents even further. Although the application of these advanced reproductive technolo- gies can be viewed as a threat to genetic diversity, they also provide powerful tools for its preservation. Germplasm from endangered breeds, unpopular lines, or populations that are being replaced by other stocks (continued on page 90)

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New Technology and Its Impact on Conservation / 89 the minor livestock species, the efficiency of cryopreservation is either less effective or unknown (Graham et al., 1984~. (See Appendix C.) The frozen semen pool can be used in breeding live conserved stocks to mitigate the effects of natural selection, inbreeding, and genetic drift. A drawback of a semen store by itself is that purebred stock can be produced only after a series of backcrosses to current stocks. The availability of frozen embryos removes this constraint, These twins were the third set of their type produced at the International Laboratory for Research on Animal Diseases using embryo transfer technology. A trypanotolerant N'Dama embryo and a trypanosensitive Boran embryo were implanted at the same time into this recipient Boran cow to produce hematopoietic chimeric twins. As they developed in the uterus, their blood was mixed through the placenta, which may result in eachfetus taking on each other's blood characteristics. Scientists at the laboratory will challenge the twins with trypanosomes to determine which, if any, trypanotolerant traits have been transferred from the N'Dama to the Boran. Credit: Inter- national Laboratory for Research on Animal Diseases.

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90 / Livestock for then the purebred stock can be produced immediately and main- tained subsequently by the more extensive pool of frozen semen. Thus the ideal package for cryopreservation is a small embryo store (100 to 200 embryos) combined with a semen store. The first cryopreservation of mammalian embryos followed by live birth was reported in mice (Whittingham et al., 1972~. Since then, embryos of about 20 mammalian species have been successfully can be retained indefinitely through judicious sampling and cryopreser- vation of semen and embryos. The capacity to regenerate commercial stocks can also be preserved through the long-term storage of semen and embryos. Studies have amply demonstrated that cryopreservation is genetically sound and provides cost-effective insurance against loss of genetic diversity (Smith, 1984a,b). This strategy is most feasible in cattle and sheep, where use of frozen semen and embryos is most com- mon, but it will be more difficult in swine and poultry, where embryos cannot be frozen and use of frozen semen is less common. Studies on the cryobiology of the latter species thus become a priority. Similar arguments can be made for the potential impact of molecu- lar biology, which allows identification of genetic diversity at the mo- lecular level. Its impact on commercial livestock populations cannot yet be predicted. However, the possibility that future commercial stocks will contain many genetically altered individuals must be considered. Depending on the source of the introduced genetic material, these ani- mals could represent an enhancement of genetic diversity through the introduction of novel genes to the species. It seems more likely, how- ever, that losses in overall genetic diversity could occur if commercial populations are founded from a limited sample of genetically manipu- lated individuals. Molecular technologies can also be used to enhance conservation efforts. Animals selected for conservation can be documented at the molecular level to possess desired genetic characteristics. DNA librar- ies, which possess cloned fragments of an animal's genome, can also preserve the genetic material of endangered stock for future use or study. However, the present inability to directly use DNA in these libraries for improving livestock means that they should not be seen as a substitute for proven methods of conservation. Recognition of the need to conserve genetic diversity must become an integral part of genetic improvement programs. The livestock breeder and molecular geneticist must be free to continue to attempt to maxi- mize rates of improvement in livestock species, secure in the knowl- edge that the same technologies that facilitate improvement efforts can also aid in securing the levels of genetic diversity required to meet unforeseen future needs.

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New Technology and Its Impact on Conservation / 91 cryopreserved, and the procedures are now routine in cattle, goats, sheep, rabbits, and mice (Leibo, 1986~. Pig embryos have been suc- cessfully frozen at expanded blastocyst (entire zone pellucida) and early hatched stages, but pregnancy rates are very low (Hayashi et al., 1989~. Three mouse embryo banks have been established for pre- serving defined inbred and mutant strains of laboratory mice (Na- tional Institutes of Health, Bethesda, Maryland; Jackson Laboratories, Bar Harbor, Maine; and Medical Research Council Laboratories, Carshalton, England). Vitrification is another promising advance in embryo cryopreser- vation. The process involves rapid cooling to transform cellular liq- uids into a glass-like viscous solid without any ice or crystal forma- tion. Success with embryos requires the use of a highly concentrated, yet effectively nontoxic solution of cryoprotectants as well as rapid cooling and warming rates (Rail and Fahy, 1985a,b). A vitrification procedure for bovine blastocysts has been developed by Van der Zwalmen et al. (1989~. Multiple Ovulation ant! Embryo Transfer An important aspect of the use of embryos has been the develop- ment of hormonal treatments leading to greater numbers of embryos per donor female. The administration of a follicle-stimulating hor- mone will cause multiple ovulations. After estrus is induced, the donor female is inseminated. Embryos are usually obtained from large animals, such as cattle, buffalo, and horses, nonsurgically while those from other species are collected surgically. Yields are variable. In cattle, six to eight embryos are obtained during the procedure. Pregnancy rates of 60 to 70 percent with fresh embryos and of 40 to 50 percent with frozen embryos have been reported. These rates, however, have been achieved and are slowly improving after sub- stantial research, development, and experience with a few breeds. Results for other breeds and species are variable, and further specific research will be required to improve methods (see Appendix B). A new series of technologies, again mainly in cattle, involves collection and maturation of oocytes (unfertilized ova) and in vitro fertilization. Oocytes may be aspirated from the ovary of a live ani- mal or excised from the ovary immediately after slaughter. The oo- cytes are then matured and fertilized in an artificial environment, and they produce normal embryos to be transplanted into recipients. These techniques are still in the developmental stage, but they will increase the potential genetic contribution of individual females to the population.

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92 / Livestock Embryo Splitting and Cloning Cloning the asexual reproduction of identical genotypes has been achieved in cattle by microsurgically splitting embryos or by transferring individual nuclei derived from multicellular embryos into enucleated oocytes (oocytes with the nucleus removed). Experience irk splitting and cloning embryos has been mainly with cattle and sheep (First, 1991~. Small groups of identical individuals have been obtained. Embryos can be mechanically divided to produce genetically identical embryos; the splitting is usually done at the 60-cell (morula) to 250- cell (blastocyst) stage. Embryos derived from embryo splitting have a lower chance of survival (for example, 50 percent versus 70 percent for intact embryos) when transferred to females. Survival of split embryos is further reduced after freezing, so splitting may not be an advantage in preservation. Embryos derived from the splitting of an original founder embryo may themselves be split to produce addi- tional identical descendants, but the survival rate of the resulting embryos is markedly reduced. Cloning by nuclear transplantation, originally developed for the mouse (McGrath and Softer, 1983), has produced viable embryos and offspring in cattle, pigs, rabbits, and sheep (First, 1991; Matter, 1992~. Using embryo transfer technol- ogy, a bovine embryo is split before implantation in a foster Boran mother to produce N'Dama-Boran crossbred calves. The first crossbred calves were produced in 1990; by the end of the year, 75 such calves were born. Large families of these cattle are needed for research at the International Laboratory for Research on Animal Diseases to locate the genes responsible for trypanotolerance, an ability of N'Dama and other indigenous livestock breeds in Africa to re- main productive while infected with trypanosomes. Credit: International Laboratory for Research on Animal Diseases.

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New Technology and Its Impact on Conservation / 93 It is based on a technique for nuclear transfer first reported in the frog, Rana pipiens (Briggs and King, 1952~. Nuclear transplantation involves the transfer of a nucleus or blastomere from a valuable em- bryo into a suitably receptive enucleated oocyte. After the reconsti- tuted egg develops to a multicellular stage, it can be cloned again. Theoretically, large numbers of identical individuals could be pro- duced using this method. Survival rates are still low, however, and the process has only been done through three generations. For conservation it is important to retain as much genetic diver- sity as possible, which is accomplished by storing unrelated geno- types. Cloning does not increase the amount of genetic diversity; rather, it enables evaluation of stored samples without reducing the amount of diversity still preserved. It can also be used to provide a back-up collection or samples for multiple users. Clones would be of value, for example, in testing genetically identical individuals across different environments. In any stock, live or cryopreserved, cloning would allow the imme- diate commercial exploitation of identified superior genotypes. Cloning has two drawbacks, however. First, only embryos have been cloned thus far, so duplicate (or multiple) frozen embryos must be retained until the clone has been evaluated for economic merit and then re- generated and multiplied. Second, the clone itself has no genetic variation and cannot be improved further. For continuous genetic improvement, the best clones would have to be mated to obtain new genetic combinations for another round of clonal selection. Determining Sex It is not yet possible to separate X-chromosome-bearing (female) and Y-chromosome-bearing (male) sperm and maintain normal sperm viability. Some progress has been made through flow cytometry, a method for rapidly measuring cells, based on the difference in the DNA content of X and Y sperm (Gledhill et al., 1982; Pinkel et al., 1985~. Flow cytometric procedures can also be used for sorting sperm cells to produce samples enriched in X or Y sperm (Iohnson et al., 1987~. These sorted samples have been used successfully to produce live offspring (Iohnson et al., 1989~. The sex of embryos can be deter- mined cytogenically from 3 or 4 cells taken at the 16- to 32-cell stage. More recently, male-specific antigen has been used to determine sex in embryos from the 16-cell to early blastocyst (100-cell) stages. DNA probes have been used to determine sex at all stages, and are avail- able commercially in Australia, France, and the United States (Herr et al., 1990; Nibart et al., 1992~.

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94 / Livestock Sexing of sperm or embryos, however, seems to be of little advan- tage in conservation. Storage costs are likely to be low relative to the costs of collection, so all sperm and embryos collected should be retained; determining the sex would only increase costs and losses in viability. If reasonable numbers of embryos are collected (for ex- ample, 100 to 200 to allow for losses), deviations from the 50:50 sex ratio are likely to be small. If very limited numbers of embryos are available, however, sexing may be used to decrease the chance of random genetic drift upon restoration of the population. HEALTH STATUS OF GERMPLASM One of the main limitations on the movement and use of genetic resources has been concern about the health status of the germplasm. The adverse effects of any imported diseases could negate potential gains from imported germplasm and deleteriously affect existing stocks. This concern has often barred exports and imports. Where transfer of germplasm has been allowed, stringent regulations and conditions have frequently been imposed. They usually involve expensive quar- antine procedures over a period of time and a series of tests in both the exporting and importing countries for a specific set of diseases. Thus, the exchange of improved genetic stocks across countries has been expensive and often restricted in the past. Improved methods of disease detection and control and the use of frozen semen and embryos make international exchange of improved genetic stocks in- creasingly feasible. However, the potential to transmit disease agents must not be minimized or overlooked (see Appendixes B and C). The preservation of rare stocks is more difficult. Limited num- bers of individuals may be available as a source of germplasm, and they are unlikely to be kept under sanitary isolation conditions. Ac- cess to the animals may be limited in number and time, and their health status may be variable. The conditions under which the ani- mals are kept and samples are collected may be marginal. Another drawback is that the preserved stocks are retained for future use. Even if they satisfy today's health regulations, they may not satisfy those prevailing in the future. In this case all effort at conservation might be in vain. Alternatively, a new generation of the preserved stock would have to be produced and placed in quarantine until the new set of health conditions could be satisfied. Thus, it is important to separate animal germplasm from disease agents at the time of storage, If possible. One advantage of preserved germplasm would be that it will be free of any diseases that have developed or spread since its collection.

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New Technology and Its Impact on Conservation / 95 New technologies of semen screening (see Appendix C) and em- bryo washing (see Appendix B) hold considerable promise for im- proving the health status of germplasm. A method developed by Schultz et al. (1982) allows verification of the health status of semen samples. This technique may be valuable for testing semen from males known to be exposed to or infected with a disease. Semen immunoextension involves the treating of semen with specific anti- bodies to prevent disease transmission. Embryo washing and treat- ment remove or neutralize pathogens that may be found around the embryo in a layer called the zone pellucida. It has been effective against most pathogens tested to date (Singh, 1987~. (See Appendix B.) These techniques have important implications for genetic conser- vation because they could allow collection of germplasm under ordi- nary farm conditions and the future testing of stored ~ermDlasm to meet new health standards. RECOMMENDATIONS ~r~~~~~ -~ Selection and crossbreeding have been used to make major con- tributions to animal improvement programs in many countries. Relatively little effort has been devoted, however, to the preservation of germplasm, which represents the biodiversity on which selection and genetic com- binations are based. Research on technologies that could benefit preservation and use of animal genetic resources should be continued and expanded. New ideas, research results, and techniques are continuously be- ing reported that can affect the methods and value of conservation. Research areas with the potential to enhance the preservation, man- agement, and use of animal genetic resources include the following: Methods for collecting and cryopreserving sperm and embryos from minor breeds and under adverse field conditions. Reliable technologies to detect, eradicate, or control pathogenic agents that may be harbored in semen, ova, embryos, or live animals. Improved methods for cryopreserving the sperm and embryos of swine and avian species. Techniques for establishing and maintaining genetic libraries for each livestock species. Development of genomic maps for each livestock species. Population genetics studies of molecular methods to measure relationships within and among breeds so that priority breeds for preservation, use, or development can be identified.

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96 / Livestock Expansion of the numbers of DNA markers (RFLPs and VNTRs) for livestock species. Techniques for marker-assisted selection in each major live- stock species. For selected species, DNA banks should be established to provide a potential source of specific genes forfuture livestock improvement. A DNA store is a potential future source of specific genes. From genomic libraries particular genes of interest can be cloned. Specific genes from a DNA store may some day be of value for improving livestock through the use of directed gene introgression (breeding) or across species through transgenic technologies. A DNA bank could ensure sufficient genetic material for molecular characterization of the specific genes and DNA segments of a population, inexpensively stored, which could provide researchers with the material for mo- lecular biological and gene mapping studies of that population. Al- though DNA stores may aid evaluation and use of germplasm, they should not be considered presently viable methods for conserving the genetic resources of livestock. Furthermore, the characterization of phenotypic attributes are not possible through DNA libraries. Efforts should be made to link research on preservation, management, and use of livestock germplasm to analogous efforts in otherfields of study. Many studies on the human genome are being carried out. Knowledge gained from genome mapping studies of livestock can be of use in human mapping studies, just as information from emerging human maps can aid livestock efforts. Several zoos are working to develop methods of germplasm manipulation and storage for wild animals (Office of Technology Assessment, 1985, 1987) and their efforts ben- efit from livestock studies. Gains in molecular genetics derived from the study of plants also may have applications to animals.