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Animal Science Opportunities for research advances that will improve the productivity of the livestock industries are-greater today than at any time in history. The powerful new tools of biotechnology can be applied directly to the de- velopment of knowledge about food animals and the produc- tion of biologicals to enhance livestock productivity. In addition, many of the genetic and reproductive manipu- lations that are not possible in humans, either ethically or practically, can be accomplished in farm animals. Spectacular advances in disease and parasite control and striking increases in the efficiency of converting feed- stuffs to meat, milk, and eggs can be expected within one to two decades if animal~science research is properly focused and supported. Scientific livestock breeding already has increased production dramatically since the time herds were driven across the plains to market. As recently as 1950, 22 million dairy cows in the United States were producing more than 2,400 kilograms per cow of milk annually. Now, only one-half the number of cows are producing the same total amount of milk while consuming one-third less total feed. The objective is not to produce more milk, but rather to produce more efficiently, with a reduction in feed, feedlot pollution, and animal maintenance costs. The turkey industry has been particularly successful in using quantitative genetic principles to produce big- breasted turkeys efficiently. Now the quality of these birds is controlled through the use of artificial insem- ination. In fact, 100 percent of the commercial turkey flocks in this country is replaced each year using arti- ficial insemination. Other industries are using similar, more traditional techniques to improve production in cattle, hogs, and sheep while continuing to explore the 32

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33 basic science of genetics to further understand and enhance genetic improvement. The new biotechnology methods, as tools that enhance conventional breeding methods, make possible for the first time realistic consideration of such ideas as the genetic engineering of an animal, one possessing more desirable production traits. Segments of DNA coding for desirable genes can be isolated in the laboratory, inserted into suitable DNA-carrying vectors, and trans- ferred into host animals or into a bacterial cloning system. Extra copies of the gene coding for growth hormone have already been inserted into mouse embryos, yielding offspring twice the normal size. Bovine growth hormone is now being produced in the laboratory by a bacterial cloning system that provides sufficient quanti- ties of this scarce biological for experiments on growth and milk production in cattle. Such experiments have major implications for improving productivity in farm animals. The exploitation of hybridoma technology--fusing a continuously dividing cell with one that produces anti- body--in the production of monoclonal antibodies has substantially enhanced the identification and isolation of genes and gene products as well as the production of highly specific antibody preparations for diagnostic and therapeutic uses. Scientists must be provided with funds and facilities to take immediate advantage of these and similar developments to explore gene structure, function, and regulation, and the basic physiology of livestock species. The following discussions specify the kind of research that the committee believes will result in improved animal productivity. Molecular Basis of Disease Each year the productivity of livestock and poultry in the United States is reduced by at least 20 percent because of diseases. This represents an estimated annual economic loss of $14 billion. Until recently the re- search approaches available to address disease losses in food animals have been limited. The availability of new technologies such as recombinant DNA and monoclonal anti- bodies now affords an exceptional opportunity to under- stand and control disease. The ability to isolate and clone genes that play a role in immunity is an enormous step toward eliminating

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34 certain diseases. Major advances relate to the growing body of knowledge about the genes regulating the immune response, the genes controlling the antigen components of diseases and parasites, and the ability to create hybrid- omas with immune cells that can yield highly specific monoclonal antibodies. The payoff from intensified efforts in animal disease control will probably come much more quickly than results from research in either metabolic regulation or reproduc- tion because of the solid foundations already being laid in this area for both man and animals. For example, studies at the ARS Plum Island Animal Disease Center in New York, on the molecular biology of foot and mouth dis- ease, in addition to dissecting the physical chemistry and biochemists of the virus, have led to trials of a promising vaccine based on the cloned surface protein of the virus. By the very nature of infection and disease, the bene- fits of vaccines are immediate, while applications in genetic improvement are slowed by the necessity of ana- lyzing results in subsequent generations. More impor- tantly, knowledge gained from studies of a particular viral or microbial disease and development of a vaccine often can be readily extended to many other diseases. Genes Regulating the Immune Response The strides that have been made in molecular genetics have been matched by those made in immunology. Now it is possible to combine the principles and techniques of molecular genetics and immunology to address one of the fundamental questions in biology--the nature of the re- cognition and response mechanisms in immunity. m e application of this basic knowledge can be immediately translated into means for protecting economically impor- tant animals from costly diseases. Current intensive investigations in both laboratory animals and humans are now providing an initial view of the structure and function of three classes of genes that control the expression of the immune response. These are the genes coding for the major histocompatibility complex (MHC), for mediator proteins called lymphokines, and for antibodies. Very little research to date, however, has been conducted on the immune response in food animals.

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35 There are two major white blood cell types, B and T lymphocytes, involved in the immune reaction. Viruses, bacteria, parasites, or other foreign substances contain immunologically' active macromolecules called antigens. When an infectious agent enters the body, its antigens stimulate the immune system to produce specialized pro- teins, or antibodies, that can recognize and bind to the antigens. Each unique antigen triggers both B cells and T cells: B cells produce antibody specific to that anti- gen, and T cells produce an antibodylike T cell receptor, also specific to the antigen. B cells release the anti- body into the blood, creating extracellular immunity, and T cells carry the T cell receptors on their cell sur- faces, providing cellular immunity. Exposure to additional antigen stimulates the B and T cells previously dedicated to that antigen to divide and produce their respective antigen-specific products. A vaccine is simply an antigen or set of antigens unique to a disease-causing organism that stimulates a specific immune response against the disease agent. The interaction of the T cell receptor with antigen is unique in that recognition involves proteins of the MHC that also play an important role in helping to dis- tinguish between foreign substances, which should be destroyed, and self proteins, which should not be de- stroyed. The host does not normally develop antibodies or T cells directed against its own proteins, although this does occur in certain autoimmune diseases. Major Histocompatibility Complex A family of genes located on a single chromosome codes for the MHC. In addition to recognition in the immune response, these genes are associated with the inheritance of diseases that appear to be malfunctions in the ability to dis- tinguish between self and nonself molecules. Rheumatoid arthritis, multiple sclerosis, and juvenile-onset dia- betes are examples of MHC-related diseases in humans. In animals, Marek's disease, a blood cancer in chickens, and scrapie, a disease of the central nervous system in sheep, appear to have strong relationships to the MHC. Certain strains of chickens with Marek's disease dem- onstrate a greater resistance to the development of lym- phomas, or tumors, than do others. Immunogeneticists have noted that these strains have a given MHC, or set of closely linked genes. Similarly, recent studies on scrapie indicate that different breeds of sheep exhibit

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36 different degrees of expression of the disease. me severity of the disease appears to be associated with the transmission of certain MHC gene components. Efforts should be intensified to elucidate fully the genetic makeup of the MHC in food animals and to define the role of gene products in regulating host immunity and susceptibility to disease. Direct application of the findings from such basic research will allow for the development of improved breeding programs. Specifically, breeders will be able to select for greater resistance to various diseases, thus upgrading the herd or flock and reducing the high direct or indirect costs of disease treatment and loss. Lymphocyte Hormones As part of the immune response, lymphocytes can be directed to secrete soluble peptides that stimulate or suppress antibody production, division, and similar activities in other cells. mese mediators, or hormones, are called lymphokines. In recent studies of such substances, interleukin-2 was shown to promote lymphocyte replication, which has greatly facilitated the in vitro growth and cloning of T cells. Another lympho- kine, interferon, already has found clinical application in the treatment of some cancers and viral diseases in humans, and is undergoing field trials as a preventive measure for bovine respiratory disease. Opportunities now exist to characterize lymphokines fully, isolate the genes that code for them, and clone these genes to obtain sufficient quantities of various lymphocyte hormones for the study of their immune response regulation properties. Scientists already have accomplished these steps with interferon. Current investigations of lymphokines, made possible with recom- binant-DNA technology, emphasize the potential for research directed toward molecules that regulate or potentiate the immune response. The discovery of natural mediators that could decrease animal losses caused by alterations in immunological com- petence during shipment, weaning, or other periods of stress would be of exceptional value. In these situa- tions, normal management of endemic diseases is hindered by the apparently altered immune response. Stress in livestock and avians often results in decreased reproduc- tion and growth performance, and, at times, even death of the animal. m is adds up to very large annual losses that are difficult to quantitate in actual dollars.

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37 The lymphokines, as natural immune modulators, may be extremely beneficial in influencing the ids une response. Basic research in this area is directing scientists toward development of natural products that are easily metabolized by the system. These products may ultimately be more effective and economical than synthetic pharma- ceuticals. Antibodies The genetic region that codes for antibodies is remarkable; it directs the synthesis of an antibody to virtually any foreign molecule by rearranging the DNA in the immune cell. Intensified investigation of antibodies and the T cell receptor in food animals would particularly apply to ongoing studies of a number of important livestock viral diseases such as bluetongue, malignant catarrhal fever, bovine leukemia, scrapie, pseudorabies, African swine fever, Marek's disease, and avian influenza and leukosis; bacterial diseases such as diarrhea of the newborn and mastitis; rickettsial dis- eases such as anaplasmosis; and parasitic diseases such as babesiosis. The greatest problem in combating these diseases is providing early diagnosis so that treatment can be given before economic loss occurs. There is an immediate need for antibody reagents that will clearly distinguish dis- ease-causing pathogens at an early stage of infection. In addition, increased knowledge of T cell functions will provide information about cell-associated immunity and the immune response. developed when scientists have a clear understanding of the immune response, which varies with the disease- causing organism and the species of animal involved. Furthermore, these studies must be conducted in food animals. Information cannot be extrapolated directly from humans or laboratory animals. Advances in the study of antibodies and the immune response in these animals will directly benefit the livestock industries and pro- vide additional benefit to medical science. Effective vaccines can best be Pathogens and Vectors Most vaccines consist of the organism that causes the disease, either killed or treated in various ways (attenuated) to reduce its virulence. m e immune system responds to the killed or attenuated vaccine by producing antibodies that bind to antigens on the surface of the pathogen, labeling it for attack. The antibodies pro-

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38 duced against the modified pathogen circulate throughout the body and render the animal resistant to a later infection by the live pathogen, thus protecting the animal against the disease. Because these vaccines contain the entire pathogen and its complete genetic material, there is some risk that attenuated strains may yet be potent enough to actually cause disease. In other cases, however, vaccines con- sisting of inactivated virus have not stimulated antibody production in the animal, and immunity has not been con- ferred. In addition, these vaccines are specific for a particular pathogen and generally offer no protection against the variety of subtly different strains that may be present. Conventional vaccines of denatured, inactivated virus have failed to provide immunity against diseases such as bovine viral diarrhea. Live vaccines have been found not only to be inadequate but also in some instances to have contributed to the spread of disease. The development of subunit vaccines, which contain only the critical part of the pathogen necessary to stimulate antibody production and not its genetic material, will solve many of the problems presented by conventional vaccines. Using monoclonal antibodies, scientists at the ARS Plum Island Animal Disease Center and others have identified and cloned the gene that codes for a major foot and mouth disease viral surface pro- tein. A subunit vaccine for one type of the virus has been produced. Foot and mouth disease attacks all cloven hooved animals, and, although it was eradicated from the United States in 1929, outbreaks in other parts of the world and the potential for transmission of the disease are continuing threats to U.S. livestock. Monoclonal antibodies give scientists the precision to completely define the virus and its strains, and aid in the genetic engineering of effective subunit vaccines. Once perfected, these steps can be applied to other viruses and to bacteria and parasites. Disease-causing parasites, including both the single- celled protozoans and the many-celled metazoans, are particularly difficult to combat with vaccines, because they have the chameleonlike ability either to alter or to mask their antigens, and thus escape recognition by the antibody. A well-known example of this phenomenon is the African trypanosomes that cause trypanosomiasis, or sleeping sickness, in humans and other animals. During an infection new antigenic variants are unaffected by the -

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39 immunity to previous variants. By the time the response to the new antigen reaches effective levels, a still newer variant is being produced. This mechanism keeps the parasite a step ahead of the host's protective response and allows its survival regardless of the effec- tiveness of the immunity. A costly example of this type of phenomenon is ana- plasmosis, the tickborne rickettsial disease that causes severe anemia and death in cattle. Anaplasmosis-related losses, including death, persistence of the infectious agent in surviving animals, and reduced performance of survivors, cost the U.S. industry $100 million in 1983. The complexity of the immune mechanism in parasitic diseases renders a disease-control program via vaccine difficult and may call for research directed in a related area--vectors. Insects and other arthropod vectors not only transmit disease but also serve as reservoirs for pathogens between disease outbreaks. Diseases can often be controlled, however, if the vector can be altered or eliminated. Cloning of specific genes of vectors--gnats, ticks, black flies and mosquitoes--can substantially increase understanding of the transmissibility of a disease agent and aid in its eradication or control. Genetic manipulations of microbial agents such as bacteria, viruses, protozoa, or fungi may result in the creation or enhancement of agents lethal to the vector. To reduce the necessity of repeated applications to vector-infested areas, a bacterial control agent, for example, would have to be genetically designed to thrive and reproduce toxin-bearing generations in the wide variety of habitats where the vectors are found. One promising use of bacterial control of vectors involves Bacillus thuringiensis, Serotype H-14, and its natural toxins that are deadly to mosquitoes and black flies. To be most effective, B. thuringiensis would have to be adapted to brackish water, pollutants, and other condi- tions common to mosquito-infested areas. Genetic studies of the characteristics of vectors should focus on factors that influence vector competence, or vector efficiency--the intrinsic factors and mecha- nisms that control the ability of insects and other arthropods to carry and transmit disease agents. Barrier systems exist in vectors that prevent a disease agent, such as a virus, from spreading into the different cells and tissues in the vector. His limits or eliminates the vector's ability to transfer the disease agent. The

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40 mechanisms of the barrier systems are not well under- stood. Hey appear to be under genetic control and can be expressed in varying degrees within a vector popula- tion, thus affecting the epidemiology of the diseases. Disease Control Extensive studies have established that many diseases can be controlled by a combination of procedures includ- ing vaccination, enhancement of the immune response, vector control, diagnosis, and therapy. As discussed previously a better understanding of infectious agents will lead to improved vaccines. Characterization of recognition properties between vectors and the disease agents they transmit will provide clues to control surest The increasing knowledge about genes that regulate the immune response has already led to the identification of lymphokines and other immune response enhancers. In addition to these areas the further exploitation of monoclonal antibodies and recombinant- DNA technology will improve current methods used to identify and control infectious agents. Molecular Diagnostics Both monoclonal antibodies and DNA manipulation can be employed to fully character- ize and detect pathogens. Monoclonal antibodies that recognize specific antigens can be prepared relatively easily in the laboratory, and pathogen-specific nucleic acid sequences can be identified by restriction enzyme mapping and by RNA or DNA hybridization techniques. me sensitivity and utility of the methods are attested to by their increased use in diagnostic research in humans. Conventional diagnostic reagents have proven inade- quate for numerous diseases. No effective reagents yet exist to diagnose malignant catarrhal fever, a fatal herpesvirus in cattle and sheep. As a consequence, there is currently no effective way to control the disease. Scrapie in sheep, introduced into the United States in 1947, is another critical disease. Scrapie presents a difficult diagnostic problem because of its extended incubation period of up to three years. Diagnostic tests based on monoclonal antibodies are under development for bovine leukosis virus and bluetongue, a viral disease in sheep that is transmitted by gnats. Monoclonal antibody diagnostic products could be useful for disease control programs both in the United

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41 States and abroad in monitoring levels of disease in a herd as well as in initially detecting disease. Mono- clonal antibodies and recombinant-DNA techniques both could be used to identify critical immunogenic components for inclusion in subunit vaccines and as tools to isolate antigens that have the potential for use as effective vaccines. Subunit vaccines also could be prepared by chemically synthesizing peptides, linear polymers of amino acids. The synthetic peptides would be based on known amino acid sequences of viral surface proteins. Synthetic peptides corresponding to part of one viral surface protein of foot and mouth disease have been shown to protect animals against live foot and mouth disease virus of that type. Subunit vaccines would provide greater effectiveness with less risk than conventional vaccines and have the potential to be produced economically. Therapeutic Agents The potential exists to use monoclonal antibodies to develop immunotherapeutic agents. In humans, antibodies to specific toxins and pathogens such as antivenin and diphtheria antitoxin are effective. Antibodies administered by mouth or by injection have recently been shown to have a beneficial effect in animals with bovine diarrhea caused by the bacteria Escherichia colt. Monoclonal antibodies increase the precision of specificity of the therapeutic agents used and avoid the problem of injecting extraneous proteins during therapy. In a number of instances the depression of the immune response results in onset of disease, for instance, in gram negative bacterial septicemia. m e use of monoclonal antibodies can potentially prevent or arrest such infections, especially where drug therapy is contra- indicated. me value of such immunotherapy has been recently demonstrated by the successful treatment of neonatal pigs and calves with monoclonal antibodies specific for pill antigens on enterotoxigenic E. cold that cause diarrhea. The whiskerlike pill, located on the surface of the bacteria, provide a means of adhesion so that the pathogen can colonize the gut mucosa and produce a high concentration of its toxin. The process is inhibited by administration of the monoclonal antibody. Research Status The immediate opportunities provided by the newer biological technologies set the stage for relatively

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42 rapid research advances in the area of animal disease. With intensive research effort and use of molecular techniques, safer and more effective vaccines and diagnostic and therapeutic products could be available within a few years. Recent appointments of new scientists trained in molecular biology at many of the ARS centers are increasing the potential productivity of each of these laboratories. Additional appointments in molecular biology, immunology, genetics, biochemistry, molecular pathology, and computer science will enhance the cata- lytic effect that these newer technologies can have on research productivity. Excellent examples of this ap- proach are the basic research program in parasitology at the Beltsville Agricultural Research Center in Maryland and the exotic animal disease research program at the Plum Island center in New York, where genetic engineering methods are being exploited to develop a safe vaccine for one type of foot and mouth disease. The ARS will benefit by focusing research on a number of specific diseases and by concentrating on the full utilization of existing Facilities and the acquisition of new equipment. Major benefits will result from an ex- tension of interdisciplinary collaboration that includes not only ARS scientists but also those from university and industrial laboratories. Special attention must be directed toward the following areas: Studies of the molecular structure of genes that regulate the immune response, the immune response itself, and the genetic basis of disease susceptibility. These areas could be pursued most efficiently at Ames, Iowa; Beltsville, Maryland; and Clay Center, Nebraska. Definition of the molecular organization and antigenic composition of pathogens including viruses, bacteria, and protozoan and metozoan parasites, to be used to develop refined molecular diagnostic tests using monoclonal antibodies. Such studies should be emphasized at the Ames and Plum Island centers. ~ Development of the scientific base for subunit vaccine production and the use of other antigenic com- ponents for the production of improved vaccines at Ames. Development of the scientific expertise to use host-derived immune modulators to enhance the immune response to improved vaccines. These might include lymphokines, such as interferons, interleukins, and others. Ames and Beltsville are appropriate centers to support research in this area.

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43 Several key laboratories around the world are contrib- uting to molecular and other aspects of research in food animal diseases. These include both government-operated and university laboratories . Molecular Basis for Metabolic Regulation The efficiency of food production by agriculturally important animals is related to the regulation of metabolism during pregnancy, growth, and lactation. The new biotechnology offers versatile approaches to the understanding of physiological processes and the sub- sequent improvement of metabolic efficiency. Already on the horizon is the promise of increases in efficiency equal to those obtained during the last 20 years using traditional animal breeding and selection programs. With perhaps the exception of the major hormones gov- erning the reproductive cycle in mammals, little is known of endocrine control in food-producing animals or of the environmental and physiological factors that alter the secretion and clearance rates of hormones. Similarly, the synergisms and antagonisms among hormones and the relationships between hormonal response and cellular receptors are unknown. Progress in understanding endocrine control mechanisms can be accelerated by using recombinant-DNA methods and monoclonal antibodies in combination with such classical techniques as electron microscopy, radioimmunoassay, and cell culture methods. Endogenous chemical mediators as well as their effects on the metabolism and function of different cell types must be identified and characterized. Research focused on understanding the influences of endogenous chemical mediators and on the consumption, digestion, and utili- zation of nutrients will lead to increased metabolic efficiency in food animals. Characterization of Endogenous Chemical Mediators Hormones are chemical mediators that coordinate body processes. During pregnancy, for example, hormones promote the uptake and use of nutrients by the gravid uterus and alter the metabolism of maternal tissues to support fetal development. Hormones likely to hold key roles in the regulation of nutrient utilization include prolactin, progesterone, estrogen, placental lactogen,

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44 glucagon, growth hormone, insulin, and corticoids. Undoubtedly, additional hormones will be identified. A1- ready the use of standard cell culture techniques has led to identification of a series of small polypeptide hor- mones such as somatomedins and epidermal and bone growth factors that may be important regulators of cellular growth. Once a peptide hormone is identified and purified, it should be possible to produce monoclonal antibodies that are specific for that hormone. Using these antibodies as probes, the location and endogenous levels of the hormone can be determined. Identification and isolation of genes that code for a particular hormone also may be possible using monoclonal antibodies or other techniques. In fact, a peptide hormone with a known amino acid sequence can be synthesized if it is of a manageable size. Suf- ficient quantities of peptide hormones might be produced in bacterial systems using recombinant-DNA techniques to permit the characterization of their biological impor- tance in food-producing animals. The potential signifi- cance is illustrated by the progress in growth hormone research. Recently, recombinantly derived bovine growth hormone has been produced in quantities large enough to administer to test animals. Preliminary results show that injections of bovine growth hormone can cause up to a 40 percent increase in milk production in dairy cows and a marked increase in growth rate in beef animals. Research in the area of endogenous chemical mediators has tremendous potential for direct applications that will result in significant increases in the efficiency of animal production. Metabolic Control and Function of Cells An elaborate system exists within the cell to regulate the metabolism of proteins, carbohydrates, and lipids. Although limited, data on food animals have frequently demonstrated critical differences among these species and laboratory animals. For example, the effects of insulin on biochemical pathways and the regulation of lipid syn- thesis in food animals have been shown to differ signifi- cantly from effects in laboratory animals and humans. Before progress can be made in many facets of cellular metabolism research, the bioregulatory processes must be characterized in key tissues such as muscle, mammary, liver, adipose, bone, and placenta. Important areas of

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45 research include the identification of nutrient and ion transport mechanisms, cellular membrane and organelle roles, and key enzymatic sites of regulation. It is critically important to identify the mechanisms by which extracellular signals, the communication between organs, arrive at an individual cell, bind to it, and then are amplified within the cell to coordinate intracellular biochemical processes. Monoclonal antibodies, affinity chromatography, and nuclear magnetic resonance, which makes use of the absorption of electromagnetic waves to identify receptor structures and characterize biochemical events, provide an unprecedented opportunity to probe the biological processes in cell metabolism. Monoclonal antibodies, for example, because of their elegant speci- ficity, can be used to block specific transport systems, enzymes, and regulatory proteins. This allows for the identification of the key steps regulating both nutrient uptake by the cell and nutrient use in such processes as protein and fat accretion in muscle and adipose tissues. A clearer understanding of these biological processes will lead to means of manipulating them to achieve great- For example, a decrease in protein turnover might markedly enhance the efficiency of muscle growth, since the rate of protein degradation is as high as 75 percent the rate of protein synthesis. Similarly, a decrease in fat deposition by adipose tissue would dramatically increase the efficiency of growth. More than 1 billion kilograms of excess fat are trimmed from beef carcasses in the United States annually, represent- ing a billion-dollar loss that is absorbed by producers, processors, and consumers. ~ er animal efficiency. Research emphasis snoula He directed toward an understanding of the basic biology that determines the partitioning between tissues such as muscle and adipose. Factors Influencing Intake and Digestion me performance of an animal is dependent upon the interactions of food consumption, digestion, and ab- sorption. Animal production is dependent upon nutrient supply and therefore upon the appetite of the animal. me control of food intake is coordinated by the central nervous system in response to gut pressure, pattern and quantity of absorbed nutrients, and factors associated with rates of tissue metabolism. Genetically superior animals that have high rates of growth or milk production

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46 are able to consume a much greater than normal quantity of feed. Conversely, loss of appetite exacerbates many of the effects of stress and clinical or subclinical disease states in animals, including humans. Unfortu- nately the biology of appetite control is not understood well enough to allow manipulation for improved production. Recent advances in high-resolution instrumentation and the use of monoclonal antibodies to identify biological mediators such as gut hormones now provide opportunities to gain new insights into the mechanisms of factors that determine food intake. There also are distinct differences among ruminant and nonruminant animals that have significant implications for production efficiency. The ability of the ruminant to utilize forages is dependent upon microbial fermen- tation. Until recently microbial action in the large intestine of nonruminants such as pigs was not fully appreciated in relation to nutrient digestion and absorption. me digestive action of these microorganisms makes possible the uptake by animals of some nutrients in feedstuffs that otherwise would not contribute to the human food supply. Genetic engineering technology makes possible the modification of organisms that might enhance the utilization of nutrients and the nutrient profile of plant materials. Microorganisms engineered to degrade plant lignin, for example, would increase the availabil- ity of nutrients from low-quality plant materials. The metabolic regulation of nutrient utilization for physiological processes such as growth and lactation is complex. Developments in biotechnology offer unique opportunities to identify and manipulate the key controls of metabolic regulation. There can be no doubt that these efforts will lead to tremendous increases in the efficiency of food production in food animals. Research Status The efficiency of food production by animals is close- ly related to the regulation of metabolism during preg- nancy, growth, lactation, and egg production. The new biological techniques, as tools, provide tremendous opportunities to understand physiological processes and to apply this knowledge to improved metabolic efficiency. The area of metabolic research within the ARS is sig- nificantly understaffed relative to its importance. An increasing number of scientists must direct their efforts

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47 to the study of growth, lactation, and reproduction in dairy and beef cattle, sheep, pigs, chickens, and tur- keys. The opportunity to create the appropriate critical mass of scientists for effective research in the basic biology of food animals will be lost unless there is con- siderable expansion or consolidation of research groups at the Beltsville Agricultural Research Center, the research center at Athens, Georgia; and the U.S. Meat Animal Research Center at Clay Center, Nebraska. The new biology methods offer unprecedented opportu- nities to probe the biological processes of cellular metabolism. The committee recommends that the Beltsville laboratories intensify their focus on basic cell biology research, capitalizing on the strong basic biomedical research programs in metabolic regulation at the neigh- boring National Institutes of Health. In addition, the ARS can further improve studies of metabolic regulation by establishing carefully focused programs at Clay Center in embryo survival, the genetic bases of disease and growth efficiency, systems modeling, and the introduction of new germ plasm. More specifically the ARS should: Identify, isolate, and characterize specific endog- enous chemical mediators involved in organ-organ and cell-cell communication; Develop fundamental knowledge of intracellular regulation of metabolism and functional interrelation- ships between organelles and other cellular components; Delineate the response mechanisms involved in the translation of extracellular signals into intracellular biochemical events; o Identify interrelationships between feedstuffs, microbial fermentation, and nutrient availability in the digestive tract; Characterize mechanisms and factors associated with the efficiency of nutrient absorption from the digestive tract; and Using this new knowledge, develop means to manipu- late these key control systems in specific tissues such as muscle, adipose, and bone, and thereby increase the efficiency of animal production. Currently only a very few small laboratory groups are studying endogenous chemical regulators and cellular metabolism.

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48 Developmental Biology and Reproduction Animals~expend an enormous amount of energy to re- produce themselves, and successful reproduction obviously is necessary to obtain sufficient animals for production purposes. Modest improvements in reproductive efficiency of livestock in this country would be worth millions of dollars annually. me new biology methods offer special opportunities to understand reproduction, which in turn should result in marked gains in productive efficiency. In addition, new tools have become available to study and modify differentiation. These tools will be of great importance in all areas of biology. Differentiation At the two-cell stage of mammalian embryonic develop- ment, each cell is equivalent and totipotent: Each cell can develop into an adult organism, resulting in identi- cal twins. As embryonic development proceeds, cells differentiate into specialized tissues, such as muscle, bone, and nerves; and totipotency is lost. No longer can a fetus be obtained from a differentiated cell such as a nerve or muscle cell. The genetic and molecular processes by which embryonic cells become specialized and then irreversibly become fixed as specific cell types are the basis for the unanswered questions of developmental biology. Is differentiation mediated primarily by changes in cytoplasm that regulate DNA or is there some fundamental change in the DNA? What is the nature of the change in cytoplasm or DNA, and is it reversible? Is the genetic information obtained via the ovum equivalent to that obtained via the sperm, and if not, how does it differ? What are the molecular mechanisms of cell-cell interaction during differentiation? Procedures incorporating nuclear transplantation and recombinant-DNA technology now provide the tools neces- sary to address these kinds of questions. Information gained in attempts to answer these questions should be useful for turning genes on and off in both cell lines and adult tissue. Just as medical researchers have switched on the gene for fetal hemoglobin production in humans with sickle cell disease to compensate for the production of defective hemoglobin by the adult gene, so too might the gene for double muscling in cattle be transferred and switched on in market animals.

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49 The double muscling mutation occurs in a number of breeds, such as the Belgian Blue; however, the animals often do not reproduce well. Manipulation of the gene in beef cattle at the appropriate stages of growth could greatly enhance productive efficiency. Understanding differentiation is absolutely fundamen- tal in nearly every area of biology; all cells, whether adipose cells or muscle cells, emerge from undifferen- tiated cells. Basic studies on differentiation should be a high priority, particularly because the tools now available will allow rapid progress. In Vitro Manipulation of Gametes and Embryos It is possible to collect embryos from females, cul- ture them in vitro, freeze and store them indefinitely in liquid nitrogen, then bring them back to activity, sex them, and transfer them back into the reproductive tract of recipients to obtain normal offspring. Individual _ _ embryos can be divided into two microsurgically, which results in identical twins. Division into three or four parts produces identical multiplets, but the success rate is lower than for twins. These techniques are useful in increasing the repro- ductive rates of females, much like artificial insemina- tion has been used in males. It is unlikely, however, that embryo transfer will replace artificial insemination on a routine production basis within the next decade, simply because of the ease and results gained using artificial insemination. It is not unusual to obtain 10,000 offspring from one bull in a single year by artificial insemination; embryo transfer might provide 15 offspring from a single cow in the same time period. Only 4 percent of the U.S. beef herd is artificially inseminated, but the technique is used in about 70 percent of the national dairy herd where the specific trait of milk production is passed successfully to subsequent generations. In the dairy industry, however, production of bull calves by embryo transfer may provide an efficient means of amplifying the genes of the best cows through their sons. Additional applications of this technology include the intercontinental transport of germ plasm via embryos economically and with less risk of spreading disease than with transport of animals or semen. The use of embryo transfer will likely increase dramatically, particularly

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so in animals of high value, as nonsurgical methods quickly replace surgical procedures and reduce the cost of equip- ment and personnel. Embryo transfer techniques also are important for basic research. For example, when genetic variation must be controlled precisely in an experiment, manufactured identical twins or multiplets can be used. Maternal effects on development can be investigated by placing half an embryo into one kind of female and the matching identical half into another. Further research, however, must be conducted to im- prove some of these methods for use in domestic animals. In vitro fertilization techniques have been particularly successful in the rabbit, mouse, and human, but work poorly in food animal species. Embryos cannot be cul- tured in vitro for longer than a day without damage, and cryopreservation kills one-fourth of the embryos. These problems are related both to species differences and to the specific technical procedures required for various animals. In the cow, for example, embryo trans- fer is a relatively simple and successful procedure, but in vitro fertilization attempts have failed. For a yet unknown reason, bovine embryos survive cryopreservation at a much higher rate than do pig embryos. In general, sperm are much easier to freeze than liver or embryo cells. There is an urgent need to conduct fundamental research in areas such as cryopreservation, in vitro fer- tilization, and nutrient requirements of embryos. mese technologies are essential for the conduct of progressive research. They would improve methods of germ plasm pres- ervation and provide insights into problems such as fer- tilization failure and embryonic death in viva. Addition of Genetic Information to Embryos The ability to obtain embryos by in vitro fertiliza- tion or to remove them from the female reproductive tract temporarily for various procedures is useful in a variety of genetic manipulations. For example, it is possible to inject genes into the pronuclei of a one-cell embryo so that the genes are duplicated automatically each time the cells divide. In this way each of the billions of cells in the resulting offspring contains the introduced gene. When rat growth hormone genes were introduced into mouse embryos by this method, the extra gene copies greatly

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51 increased the growth rate and subsequently, the size of mice. Using gene transfer techniques, useful genes could be transferred from one species into another, an accomplish- ment that would be impossible using selective mating. An example of this manipulation is the transfer of the Boorola fecundity gene from sheep to cattle. Sheep carrying this gene release four or five eggs at ovulation rather than one, a circumstance that would generally not be favored by natural selection but one that scientists might exploit to increase reproductive productivity. Cell fusion techniques might be employed to transfer genetic material from a somatic cell into a fertilized single-cell embryo for cloning. Viral vectors also might be used as another method of introducing genetic mate- rial--retroviruses may be ideal for this purpose. It should be possible to introduce new genes into the fer- tilizing sperm or the embryo itself by direct uptake of DNA from the bathing medium. Another useful research tool is the literal mixing of cells from different embryos to form chimeras. Resulting animals possess cells of different genetic composition in different parts of the body. Similar procedures have been used to create a goat-sheep chimera, known as the seep, which presents an opportunity for the study of the relationship between cells of different species during development. Clearly, these are compelling tools that can be used to answer the fundamental questions of animal reproduc- tion. mere is an immediate need to develop these emerg- ing technologies for application to livestock for the future benefit of animal production. Reproductive Efficiency Reproductive success is central to efficient animal production. me increasing economic values of growth rate efficiency and resistance to disease are magnified by improvements in the reproduction rate. Less than 70 percent of adult female farm animals produce live young in any given breeding season. Barriers to reproductive efficiency include production of nonviable gametes, fer- tilization failure, embryonic mortality, and losses at birth and in the first few weeks of extrauterine life. Two of these areas, oogenesis, the production and maturation of the egg, and embryonic mortality, are

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52 especially appropriate for study with the newly available biotechnologies and will become particularly pertinent as embryo transfer technology becomes more widely adopted. Food animals have approximately 100,000 ova in their ovaries at birth; no new ova are made after this time. In the course of a reproductive lifetime, several hundred eggs may be ovulated. More than 90 percent, however, degenerate via a process called atresia. Virtually nothing is known about control of atresia. New biotech- nologies, such as cell fusion, could be used to study the normality of the genome, thus providing a better under- standing of the nature of this process. Such investiga- tions would increase fundamental knowledge and might also result in the discovery of practical ways of harvesting large quantities of ova. Embryonic wastage is an even more serious problem; about 25 percent of all conceptions in food animals result in early embryonic death. Some embryonic wastage may be due to infectious diseases, and where the cause is unknown, pregnancy may be terminated because the embryos are genetically abnormal or because of an abnormal uter- ine environment. m e new biotechnologies such as embryo transfer provide a means of understanding the problem. It is en- tirely appropriate that agricultural research be expanded in this area, especially since species differences in reproductive processes necessitate the study of food animals themselves. Research Status The new biology methods can greatly enhance the understanding of reproduction and the study and modifi- cation of differentiation, important not only to the agricultural sciences but to all areas of biology. To establish a leadership stance in developmental and reproductive biology, the ARS must bring clear focus and depth to its existing programs by consolidating the pro- grams at a number of centers and then expanding research efforts, primarily at two centers, Beltsville and Clay Center. Major areas of research emphasis should include: . In vitro manipulation of gametes and embryos, specifically the maturation of oocytes, in vitro fertilization, and in vitro culture techniques; Addition of genetic information to gametes and embryos;

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53 Study of the genome at the molecular level; and Study of oogenesis and embryonic mortality. me committee also recommends that the ARS establish a food animal gene bank that would assist the animal science research community by facilitating, coordinating, and fostering the storage and maintenance of DNA librar- ies, gene transfer vectors, and probes. This service might be analogous to tissue culture cell banks established and maintained for the biomedical research community, including the registry of cell lines at the American Type Culture Collection, Rockville, Maryland; the Human Genetic Mutant Cell Repository maintained by the Institute for Medical Research, Camden, New Jersey; and the National Cancer Institute's Frozen Tumor Bank maintained at the Frederick Cancer Research Facility in Frederick, Maryland. There are several substantial laboratory groups in institutions studying animal differentiation, in vitro manipulation, addition of genetic information to gametes and embryos, and reproductive efficiency. With the exception of reproductive efficiency, most of this work has not been applied to food animals.