Encyclopedia Of Animal Science - T potx

Transgenic Animals: Improved Performance Vernon G Pursel United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, U.S.A INTRODUCTION With the world’s population increasing by more than 70 million people each year, modern agricultural methods that include animal biotechnology will need to be adopted if this ever-increasing population is going to avoid massive conflict over agricultural resources The ability to isolate, clone, and transfer individual genes into farm animals provides the opportunity for scientists to produce transgenic animals with modified traits that are unattainable through genetic selection This article reviews progress on transfer of genes for productivity traits into farm animals, and some areas that offer promise for the future GROWTH-RELATED TRANSGENES Early transgenic farm animal research was inspired by the dramatic growth of transgenic mice that expressed a growth hormone (GH) transgene.[1] A number of transgenic pigs and sheep were subsequently produced with human, bovine, rat, porcine, or ovine GH under the control of several gene promoters.[2] Although pigs expressing GH transgenes grew faster, utilized feed more efficiently, and were much leaner than their nontransgenic siblings, they were not larger and exhibited several notable health problems, which included lameness, susceptibility to stress, gastric ulcers, and reproductive problems.[2] The GH transgenic lambs did not grow faster or utilize feed more efficiently than control lambs, but they were much leaner and had serious health problems.[2] More recently, an insulin-like growth factor-I (IGF-I) transgene has been used to produce transgenic pigs with enhanced muscle development and reduced fat in the carcass, but the transgene did not improve growth rate or feed efficiency In contrast to the GH transgenic pigs, definitive phenotypes for the IGF-I transgenic pigs were not detected, and no gross abnormalities, pathologies, or health-related problems were encountered.[3] MODIFICATION OF MILK COMPOSITION Transfer of genes to alter milk composition has thus far received little research emphasis, but offers the dairy Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019824 Copyright D 2005 by Marcel Dekker, Inc All rights reserved industry considerable potential for the future A list of potential changes in milk components worthy of consideration is shown in Table About 80% of milk protein from cows is composed of caseins (S1, S2, and k), and whey proteins (b-lactoglobÁ ulin, a-lactalbumin, serum albumin, and g-globulin) make Á up the remaining 20% The caseins form the curds in cheese, whereas the whey proteins represent a less valuable by-product Elimination of b-lactoglobulin from milk would benefit Á cheese production because it inhibits rennin’s action on kcasein,[4] and would benefit certain fluid milk consumers because b-lactoglobulin is responsible for some milk Á allergies Removal of b-lactoglobulin from cattle is now Á technically feasible during transfection of fetal fibroblasts that are then used for nuclear transfer.[7] While removal of a-lactalbumin (a-lac) from cows’ Á Á milk may be beneficial for some consumers, researchers at the University of Illinois have shown that increased concentrations of lactose, which result from a-lac Á expression, may be beneficial for piglet growth.[8] They produced transgenic pigs that express bovine a-lactalbuÁ in their milk, which results in a higher milk lactose content in early lactation and a 20 to 50% greater milk yield on days of lactation, compared to that of control sows Weight gain of piglets suckling a-lac sows was Á greater at days and 21 after parturition than that of control piglets Thus, overexpression of a-lac milk protein Á provides a means for improving growth performance of piglets through enhanced lactation of sows WOOL PRODUCTION Three transgenic approaches have been investigated for enhancing wool production or improving wool quality The first involved transfer of bacterial genes that had the capacity to synthesize cysteine from hydrogen sulfide and serine, both of which are available in the rumen Cysteine is the rate-limiting amino acid for wool production, so an endogenous source of this amino acid has the potential to stimulate wool growth The second approach to improve wool production was to stimulate fiber growth by expression of an IGF-I transgene specifically in wool follicles The third approach was to improve wool fiber quality by altering expression of wool fiber keratin and 837 838 Transgenic Animals: Improved Performance Table Some proposed modifications of milk constituents Change Consequence Increase a and b caseins Á Á Increase phosphorylation sites in caseins Introduce proteolytic sites in caseins Increase k casein concentration Eliminate b lactoglobulin Á Decrease a lactalbumin Á Add human lactoferrin Add human lysozyme Add proteolytic sites to k casein Decrease expression of acetyl CoA carboxylase Express immunoglobulin genes Replace bovine milk protein genes with human equivalents Enhanced curd firmness for cheesemaking, improved thermal stability, and increased calcium content Increased calcium content, improved emulsification Increased rate of textural development to improve cheese ripening Enhanced stability of casein aggregates, decreased micelle size, decreased gelation and coagulation Decreased high temperature gelation, improved digestibility, decreased allergenic response, decreased primary source of cysteine in milk Decreased lactose, increased market potential of fluid milk, decreased ice crystal formation, compromised osmotic regulation of mammary gland Enhanced iron absorption, protection against gut infections Increased antimicrobial activity, reduced rennet clotting time, and increased cheese yield Increased rate of cheese ripening Decreased fat content, improved nutritional quality, reduced milk production costs Protection against pathogens such as salmonella and listeria Mimic human breast milk (Source: Refs 6.) keratin-associated protein genes in the wool follicle cortex.[9] Research on the latter approach is still underway in South Australia ENHANCED ANIMAL HEALTH Economic losses from diseases of farm animals have been estimated to amount to 10 to 20% of the total production costs Use of transgenesis in farm animals holds great promise for augmenting conventional breeding techniques to confer animals with improved resistance to these diseases and thereby reduce these losses and enhance animal welfare Unfortunately, most of the genes involved in disease resistance or susceptibility to disease are still largely unknown In addition to naturally occurring resistance genes, transgenes could be composed of genes that enhance immune response or in vitro-designed gene products (Table 2) Several approaches that have been investigated include transfer of genes for providing Table Naturally occurring disease resistance/susceptibility genes and in vitro designed genes conferring resistance Resistance Type Item Innate resistance Nonspecific immunity Specific (acquired) immunity Mechanism (Source: Ref 10.) Immunization (i.e., antibody production) Interference with pathogen entry Interference with pathogen replication Genes Controlling pathogen replication (e.g., interferons) Encoding receptors for pathogens Antimicrobial peptides Enhancing the level and type of the immune response (e.g., chemokines and cytokines) Encoding complement proteins Regulating phagocyte uptake and killing (e.g., NOS, Nramp) Encoding receptors binding directly or indirectly to antigens (T cell receptors, immunoglobulins, major histocompatibility complex, etc.) DNA vaccines, immunoglobulin cDNAs Recombinant pathogen receptors, coreceptors, etc Antisense RNA, ribozymes, intrabodies Transgenic Animals: Improved Performance resistance to influenza in pigs, preformed antibodies in pigs, viral envelope proteins in chickens and pigs, and antimicrobial peptides.[10] As a first step toward enhancing mastitis resistance of dairy animals, researchers generated transgenic mice that secrete a potent antistaphylococcal protein, lysostaphin, into milk.[11] Lysostaphin is a peptidoglycan hydrolase normally produced by Staphylococcus simulans that is active against Staphylococcus aureus bacteria S aureus is the major contagious mastitis pathogen, accounting for more than 15% of mastitis infections, and has proved difficult to control using standard management practices Three lines of transgenic mice were produced with an ovine b-lactoglobulin gene directing the secretion of Á lysostaphin into milk Progeny of these mice exhibited substantial resistance to an intramammary challenge of S aureus, with the highest expressing line being completely resistant to infection These results clearly demonstrated the potential of a transgene to combat one of the most prevalent diseases of dairy cattle The same lysostaphin transgene has now been used to produce transgenic dairy cattle that are currently being evaluated REDUCED ENVIRONMENTAL POLLUTION In an effort to reduce phosphorus excretion in swine manure, researchers at the University of Guelph[12] constructed a transgene to provide expression of phytase in salivary glands of pigs The saliva of these pigs contains the phytase enzyme that allows the pigs to digest the phosphorus in phytate, which is the most abundant source of phosphorus in the pig diet Without this enzyme, phosphorus in phytate passes undigested into feces to become the single most important pollutant of swine manure Their research showed that salivary phytase essentially provides complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75% These pigs offer a unique biological approach to the management of phosphorus nutrition and reduce one of the major environmental pollutants generated on swine farms 839 improving the efficiency of nuclear transfer, which will distinctly reduce the cost of producing transgenic cattle REFERENCES 10 11 CONCLUSION In the past few years, transgenic research to alter carcass composition, increase milk production in sows, enhance disease resistance, and reduce excretion of phosphate in pigs has shown substantial progress Modification of milk composition traits in dairy cattle offers considerable potential, but much of this research is dependent upon 12 Palmiter, R.D.; Brinster, R.L.; Hammer, R.E.; Trumbauer, M.E.; Rosenfeld, M.G.; Birnberg, N.C.; Evans, R.M Dramatic growth of mice that develop from eggs micro injected with metallothionein growth hormone fusion genes Nature 1982, 300, 611 615 Pursel, V.G.; Rexroad, C.E., Jr Status of research with transgenic farm animals J Anim Sci 1993, 71 (Suppl 3), 10 19 Pursel, V.G.; Mitchell, A.D.; Wall, R.J.; Solomon, M.B.; Coleman, M.E.; Schwartz, R.J Transgenic Research to Enhance Growth and Lean Carcass Composition in Swine In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 77 86 Jimenez Flores, R.; Richardson, T Genetic engineering of the caseins to modify the behavior of milk during processing: A review J Dairy Sci 1985, 71, 2640 2654 Yom, H C.; Bremel, R.D Genetic engineering of milk composition: Modification of milk components in lactating transgenic animals Am J Clin Nutr 1993, 58 (Suppl), 299 306 Maga, E.A.; Murray, J.D Mammary gland expression of transgenes and the potential for altering the properties of milk Bio/Technology 1995, 13, 1452 1457 Denning, C.; Burl, S.; Ainslie, A.; Bracken, J.; Dinnyes, A.; Fletcher, J.; King, T.; Ritchie, M.; Ritchie, W.A.; Rollo, M.; de Sousa, P.; Travers, A.; Wilmut, I.; Clark, A.J Deletion of the alpha (1,3) galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep Nat Biotechnol 2001, 19, 559 562 Noble, M.S.; Rodriguez Zas, S.; Cook, J.B.; Bleck, G.T.; Hurley, W.L.; Wheeler, M.B Lactational performance of first parity transgenic gilts expressing bovine alpha lactalbumin in their milk J Anim Sci 2002, 80, 1090 1096 Bawden, C.S.; McLaughlan, C.J.; Walker, S.K.; Speck, P.A.; Powell, B.C.; Huson, M.J.; Jones, L.N.; Rogers, G.E Improvement of Wool Quality by Transgenesis In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 67 76 Muller, M Increasing Disease Resistance in Transgenic ă Domestic Animals In Molecular Farming; Toutant, J.P., Balazs, E., Eds.; INRA: Paris, 2001; 87 97 Kerr, D.E.; Plaut, K.; Bramley, A.J.; Williamson, C.M.; Lax, A.J.; Moore, K.; Wells, K.D.; Wall, R.J Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice Nat Biotech nol 2001, 19, 66 70 Golovan, S.P.; Meidinger, R.G.; Ajakaiye, A.; Cottrill, M.; Wiederkehr, M.Z.; Barney, D.J.; Plante, C.; Pollard, J.W.; Fan, M.Z.; Hayes, M.A.; Laursen, J.; Hjorth, J.P.; Hacker, R.R.; Phillips, J.P.; Forsberg, C.W Pigs expressing salivary phytase produce low phosphorus manure Nat Biotechnol 2001, 19, 741 745 Transgenic Animals: Modifying the Mitochondrial Genome Carl A Pinkert University of Rochester Medical Center, Rochester, New York, U.S.A Lawrence C Smith Universite de Montreal, Quebec, Canada ´ ´ Ian A Trounce University of Melbourne, Victoria, Australia INTRODUCTION In comparison to the techniques successfully employed for nuclear gene transgenesis in livestock over the past 20 years, the lack of comparable recombination in mitochondrial DNA (mtDNA) has, until recently, prevented its direct in vivo manipulation The coordinated expression of single-copy nuclear gene products, together with the polyploid mtDNA gene products, is required for normal mitochondrial biogenesis and respiratory chain function It is of great current interest to seek improved technologies for manipulating the mitochondrial genome, so that interactions of nuclear and mtDNA genotypes can be studied in experimental systems MITOCHONDRIAL GENETICS AND ANIMAL MODELING Mammalian mitochondria contain between one and approximately ten copies of a closed, circular, supercoiled, double-stranded DNA that is bound to the inner mitochondrial membrane and is not associated with histones or a scaffolding protein matrix The mtDNAs of all vertebrates are highly conserved and quite small ($ 16.5 kb in length) in comparison to the nuclear genome Mammalian mitochondria have their own genetic systems, replete with a unique genetic code, genome structure, transcriptional and translational apparatus, and tRNAs Perhaps, because of a postulated less-extensive mitochondrial DNA (mtDNA) repair system and because of the absence of protective histones, the mitochondrial genome is subject to an increased sensitivity to mutations due to metabolic (e.g., oxidative stress) and environmental (e.g., toxins, mutagens, and UV light) sources Mitochondrial genes encode for 13 of the protein subunits that function in the mitochondrial oxidative phosphorylation 840 system, along with two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs) Accordingly, directed modification of mitochondrial genes and/or their function would provide a powerful tool in production agriculture.[1] Cytoplasmic-based traits in domestic animals have included growth, reproduction, and lactation In addition, mitochondrial restriction fragment-length polymorphisms (RFLPs) were identified and associated with specific lactational characteristics in a number of dairy cattle lineages The matrilineal inheritance of mammalian mtDNA has also been used to advantage in studies exploring the timing and geography of domestication events, as recently demonstrated for horses, where multiple domestication events appear to have occurred in the Eurasian steppe.[2] In addition, metabolic and cellular abnormalities in humans were correlated to mutations arising exclusively within the mitochondrial genome Indeed, various diseases have been associated with mtDNA point mutations, deletions, and duplications (e.g., diabetes mellitus, myocardiopathy, and retinitis pigmentosa) as well as age-associated changes in the functional integrity of mitochondria (as seen in Parkinson’s, Alzheimer’s, and Huntington’s diseases) As such, for both agricultural and biomedical research efforts, the ability to manipulate the mitochondrial genome and to regulate the expression of mitochondrial genes would provide one possible mode of genetic manipulation and therapy The creation of heteroplasmic transmitochondrial animals has developed along three lines: 1) direct mitochondrial injection into oocytes or embryos; 2) embryonic stem (ES) cell-based technologies; and 3) in relation to karyoplast or cytoplast transfer (including consequences associated with nuclear transfer or cloning experimentation; Table 1) These techniques have illustrated model systems that will provide a greater understanding of mitochondrial dynamics, leading to the development of genetically engineered production animals, and therapeutic Encyclopedia of Animal Science DOI: 10.1081/E EAS 120024365 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Transgenic Animals: Modifying the Mitochondrial Genome 841 Table Methods for creating mitochondrial modifications in animals Heteroplasmy/ homoplasmy detected Germline transmission Mitochondrial injection into ova Karyoplast fusion (nuclear transfer) Karyoplast or cytoplast transfer into ES cells and transfer Cytoplast/ooplasm transfer Heteroplasmy Heteroplasmy Yes Yes Yes Yes Heteroplasmy Yes Sperm mediated ? ? Method strategies for human metabolic diseases affected by aberrations in mitochondrial function As described in a number of recent reports,[3,4] nuclearencoded genes and knock-out modeling have been informative in identifying novel models in mitochondrial disease pathogenesis as well as critical pathways associated with mitochondrial function With initial characterization of these nuclear gene-encoded models, our search for a greater understanding of mitochondrial interactions and function would eventually lead us to a desire to develop methodology for mitochondria and mitochondrial gene transfer As a first step, efficient methods to introduce foreign or altered mtDNA or genomes into somatic or germ cells would be needed TRANSMITOCHONDRIAL ANIMALS To make a transmitochondrial animal, the ability to manipulate normal and mutant mitochondria in vivo has been a critical and difficult first step In vivo mitochondrial gene transfer remains a technological hurdle in the development of mitochondria-based genetic therapies and in the generation of experimental animal models for the study of mitochondrial dynamics and mitochondria-based traits While gene transfer has been performed in a host of cell types and organisms, transfer of nuclear DNA has been the only demonstrable form of mammalian gene transfer, short of cell fusions, to date Rapid segregation of mtDNA genotypes could occur in mammals and was first demonstrated in Holstein cattle where pedigree records in the industry allowed detailed analysis of maternally related individual genotypes.[5] Segregation of mtDNA was investigated in maternal lineages of heteroplasmic mice created by cytoplast fusion[6,7] and by embryonic karyoplast transplantation.[8] Although mitochondrial segregation in somatic tissues is Limitations Low level heteroplasmy Varying efficiencies using PEG or electrofusion Availability of germline competent/efficient cell lines Varying efficiencies and low level heteroplasmy Rare event, aberrant recombination, or programmed destruction postfertilization effective in some tissues and with increasing age, the preceding studies have shown that mtDNA heteroplasmy is maintained at stable levels throughout several generations This would suggest that the mouse germline is not very effective in segregating mtDNA haplotypes In cattle, however, highly heteroplasmic females will produce homoplasmic oocytes, whereas heteroplasmic bulls produce mostly heteroplasmic sperm, indicating that mtDNA segregation is very stringent in the female and practically absent in the male germline.[9] Together, these results suggest that mammalian species show variable patterns of mtDNA segregation In contrast to these techniques, our efforts to devise a direct mitochondria transfer technique offered certain advantages Principally, the ability to use isolated mitochondria for the production of heteroplasmic mice would allow for investigations into the feasibility of genetic manipulation of mtDNA in vitro prior to mitochondria microinjection into zygotes CONCLUSIONS Through the early 1990s, various early attempts to create transmitochondrial strains of mammalian species by introduction of foreign mitochondria into germ cells were largely unsuccessful A number of constraints have been identified or postulated, from perturbations of biological pathways to mechanistic aspects of the specific protocols used Since 1997, a number of laboratories have reported on methodologies used to create transmitochondrial animals To date, methods for mitochondria isolation and interspecific transfer of mitochondria have been reported both in laboratory and domestic animal models.[3,10,11] Interestingly, early reports on development of cloned animals by nuclear transfer resulted in conflicting consequences when retrospective studies on mitochondrial 842 transmission were reported.[12–16] Indeed, dependent upon the specific methodology employed for nuclear transfer and cytoplasm/ooplasm transfer to rescue low-quality embryos, additional models of heteroplasmy may or may not have been characterized as a consequence of mitochondrial dysfunction As such, research independent of targeted mitochondrial genomic modifications may also help unlock mechanisms underlying the dynamics related to persistence of foreign mitochondria and maintenance of heteroplasmy in various cloning protocols ARTICLES OF FURTHER INTEREST Biotechnology: Stem Cell and Germ Cell Technology, p 146 Biotechnology: Transgenic Animals, p 149 Contributions to Society: Biomedical Research Models, p 239 Genetics: Molecular, p 466 Molecular Biology: Animal, p 653 Transgenic Animals: Improved Performance, p 837 Transgenic Animals: Modifying the Mitochondrial Genome 10 11 12 REFERENCES Pinkert, C.A Genetic Engineering of Animals In Hand book of Biomedical Technology and Devices; Moore, J.E., Jr., Zouridakis, G., Eds.; CRC Press: Boca Raton, 2004; 18 18 12 Vila, C.; Leonard, J.A.; Gotherstrom, A.; Marklund, S.; Sandberg, K.; Liden, K.; Wayne, R.K.; Ellegren, H Widespread origins of domestic horse lineages Science 2001, 291 (5503), 474 477 Pinkert, C.A.; Trounce, I.A Production of transmitochon drial mice Methods 2002, 26 (4), 348 357 Wallace, D.C Mouse models for mitochondrial disease Am J Med Genet 2001, 106 (1), 71 93 Olivo, P.D.; Van de Walle, M.J.; Laipis, P.J.; Hauswirth, W.W Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D loop Nature 1983, 306 (5941), 400 402 Jenuth, J.P.; Peterson, A.C.; Fu, K.; Shoubridge, E.A Random genetic drift in the female germline explains the 13 14 15 16 rapid segregation of mammalian mitochondrial DNA Nat Genet 1996, 14 (2), 146 151 Jenuth, J.P.; Peterson, A.C.; Shoubridge, E.A Tissue specific selection for different mtDNA genotypes in heteroplasmic mice Nat Genet 1997, 16 (1), 93 95 Meirelles, F.V.; Smith, L.C Mitochondrial genotype segregation in a mouse heteroplasmic lineage produced by embryonic karyoplast transplantation Genetics 1997, 145 (2), 445 451 Smith, L.C.; Bordignon, V.; Garcia, J.M.; Meirelles, F.V Mitochondrial genotype segregation and effects during mammalian development: Applications to biotechnology Theriogenology 2000, 53 (1), 35 46 Meirelles, F.V.; Bordignon, V.; Watanabe, Y.; Watanabe, M.; Dayan, A.; Lobo, R.B.; Garcia, J.M.; Smith, L.C Compete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte Genetics 2001, 158 (1), 351 356 McKenzie, M.; Trounce, I.A.; Cassar, C.A.; Pinkert, C.A Production of homoplasmic xenomitochondrial mice Proc Natl Acad Sci USA 2004, 101 (6), 1685 1690 Hiendleder, S.; Zakhartchenko, V.; Wenigerkind, H.; Reichenbach, H.D.; Bruggerhoff, K.; Prelle, K.; Brem, G.; Stojkovic, M.; Wolf, E Heteroplasmy in bovine fetuses produced by intra and inter subspecific somatic cell nuclear transfer: Neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood Biol Reprod 2003, 68 (1), 159 166 Evans, M.J.; Gurer, C.; Loike, J.D.; Wilmut, I.; Schnieke, A.E.; Schon, E.A Mitochondrial DNA genotypes in nuclear transfer derived cloned sheep Nat Genet 1999, 23 (1), 90 93 Hiendleder, S.; Schmutz, S.M.; Erhardt, G.; Green, R.D.; Plante, Y Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle Mol Reprod Dev 1999, 54 (1), 24 31 Steinborn, R.; Schinogl, P.; Zakhartchenko, V.; Achmann, R.; Schernthaner, W.; Stojkovic, M.; Wolf, E.; Muller, M.; Brem, G Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning Nat Genet 2000, 25 (3), 255 257 Takeda, K.; Takahashi, S.; Onishi, A.; Goto, Y.; Miya zawa, A.; Imai, H Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer J Reprod Fertil 1999, 116 (2), 253 259 Transgenic Animals: Secreted Products Michael J Martin David A Dunn Carl A Pinkert University of Rochester Medical Center, Rochester, New York, U.S.A INTRODUCTION Interest in modifying traits that determine productivity of domestic animals was greatly stimulated by early experiments in which body size and growth rates were dramatically affected in transgenic mice expressing growth hormone transgenes driven by a metallothionein (MT) enhancer/promoter From that starting point, similar attempts followed to enhance growth in farm animals by introduction of various growth factors, modulators, and their receptors It soon became apparent that transgene regulation was an exquisite balancing act, where precise regulation of transgenes was crucial to normal development Yet, the overexpression of various transgene products illustrated that such animals could produce biologically important molecules as efficient mammalian bioreactors, with efficiencies far greater than conventional bacterial or cell culture systems From early studies in the mid-1980s through the 1990s, one of the main targets of genetic engineering or gene pharming efforts has involved attempts to direct expression of transgenes encoding biologically active human proteins in farm animals To date, expression of foreign genes encoding various protein products was successfully targeted to the mammary glands of goats, sheep, cattle, and swine, yet the jump from model to achieving regulatory approval has proven most challenging ADVANTAGES OF RECOMBINANT PROTEIN SYNTHESIS IN TRANSGENIC ANIMALS Several different organisms have been harnessed to produce recombinant proteins Bacteria, yeast, fungi, plants, and cultured mammalian cells can all be reprogrammed and, if properly managed, yield relatively large amounts of recombinant proteins Problems begin to arise, however, when one examines the ability of these organisms to posttranslationally modify and even release recombinant proteins Bacteria, for example, are often unable to package and secrete recombinant proteins In these instances, the recombinant protein must be physiEncyclopedia of Animal Science DOI: 10.1081/E EAS 120019825 Copyright D 2005 by Marcel Dekker, Inc All rights reserved cally extracted from the bacteria, a process that can be difficult and costly Whereas yeast can secrete recombinant proteins that are glycosylated, the enzymatic pathway(s) that they utilize to accomplish protein glycosylation differs from that employed in higher plants and animals As a result, many of the recombinant proteins produced by yeast exhibit inadequate glycosylation Posttranslational modification of recombinant proteins produced in fungi appears to be aberrant in many instances as well Mammalian cell lines, in contrast, typically perform posttranslational modifications of recombinant proteins that are quite similar to those observed in indigenous proteins Primary drawbacks to the synthesis of recombinant proteins in animal cell lines include cost and the logistical challenge associated with developing and managing cell cultures for large-scale protein production In contrast, transgenic animals, as Louis-Marie Houdebine describes,[1] share most of the properties of animal cells in culture, exhibit appropriate posttranslational modifications of recombinant proteins, and synthesize and secrete proteins extremely efficiently Indeed, mammary gland epithelia typically have a cell density that is 100- to 1000-fold greater than that used in mammalian cell culture bioreactors In one recent example, 35 transgenic goats that produced a human monoclonal antibody at a concentration of g/L in their milk were equivalent to an 8500-liter batch cell culture running 200 days/year with a g/l final production level.[2] Thus, from a production standpoint, the amount of antibody synthesized in 170,000-liter cell culture yield was equivalent to that generated in 21,000 liters of milk from transgenic goats Assuming a process yield of 60%, both systems would generate 100 kg of purified monoclonal antibody, yet the transgenic bioreactor was significantly more efficient Another obvious incentive for the production of biopharmaceuticals in transgenic livestock is their potential economic value (Table 1) The cost of human proteins obtained from donated plasma and used in replacement therapy has ranged from $4/g for serum albumin and $5000/g for antithrombin III to $150,000/g for human blood clotting factor VIII (FVIII).[4] Although the individual values of these seem dramatic, they pale in 843 844 Transgenic Animals: Secreted Products Table Molecular pharming projects: Potential biomedical and commercial products from transgenic farm animals Products a antitrypsin a proteinase inhibitor a fetoprotein (rhAFP) Use Hereditary emphysema /cystic fibrosis Hereditary emphysema /cystic fibrosis Myasthenia gravis, multiple sclerosis, and rheumatoid arthritis Antithrombin III (rhATIII) Emboli /thromboses b glucosidase Glycogen storage disease Collagen Rheumatoid arthritis CFTR Ion transport /cystic fibrosis Factor VIII Hemophilia A Factor IX Blood coagulation /hemophilia Fibrin, fibrinogen Tissue sealant development Hemoglobin Blood substitute development Lactalbumin Food additive Lactoferrin Immunomodulatory, antiinflammatory MSP (Merozoite Surface Protein 1) Malarial vaccine Phytase (EnviropigTM) Bioremediation, pollution control Human antibodies Biotherapeutics, biodefense Human C1 inhibitor Hereditary angioedema Human lysozyme Antimicrobial, immune modulator Human protein C Blood coagulation Human serum albumin Blood pressure, trauma /burn treatment Spider silk (Biosteel1) Materials development tPA Dissolve fibrin clots /heart attacks Tissues /organs Engineered for xenotransplantation Monoclonal antibodies and immunoglobulin fusion proteins: 5G1.1 Rheumatoid arthritis, nephritis AntegrenTM Neurological disorders CTLA4Ig Rheumatoid arthritis D2E7 Rheumatoid arthritis huN901 Small cell lung cancer MM 093 Myasthenia gravis, multiple sclerosis, and rheumatoid arthritis PRO 542 HIV/AIDS Crohn’s disease, rheumatoid arthritis Remicade1 Commercializing firm(s) (Bayer/PPL) (Bayer/PPL) (Merrimack/GTC) (GTC) (Pharming) (Pharming) (GTC) (ARC) (GTC, PPL) (ARC, PPL, Pharming) (Baxter) (Univ Illinois) (Pharming) (GTC) (Univ Guelph) (Abgenix, Hematech, Medarex) (Pharming) (UC Davis) (ARC, PPL) (Pharming; GTC) (Nexia) (Genzyme) (Alexion, Bresagen, Novartis) (Alexion /GTC) (Elan /GTC) (Bristol Myers Squibb/GTC) (Abbott /GTC) (ImmunoGen /GTC) (Merrimack /GTC) (Progenics /GTC) (Centocor /GTC) (Adapted from Ref 3.) comparison to the projected worth of a number of recombinant structural products Biomedical applications of BiosteelTM (Nexia Inc.), a recombinant form of dragline spider silk, produced in the milk of transgenic goats, is projected to represent $150 to $450 million in annual earnings (exclusive of military and other industrial applications) EXPRESSION OF RECOMBINANT PROTEINS IN MILK Since the introduction of the first exogenous genes into mice, more than 60 proteins have been produced in milk of transgenic animals In order to target protein expression specifically to the mammary gland, a transgene typically consists of the desired protein gene fused to one of several available mammary-specific regulatory sequences.[3–7] These sequences have included: ovine BLG; murine, rat, and rabbit whey acidic protein (WAP); bovine a-s1 casein; rat, rabbit, and goat b-casein; and guinea pig, ovine and caprine, and bovine a-lactalbumin While expression of the target protein can be achieved using either a genomic DNA or cDNA coding sequence(s), the former normally yields higher levels of protein expression Therapeutic monoclonal antibodies produced in the mammary gland of a transgenic animal line present a potentially valuable technology Transgenic monoclonal antibodies are produced by cloning genetic sequences for both heavy- and light-chain genes downstream of Transgenic Animals: Secreted Products mammary gland-specific regulatory elements Chimeric antibodies may also be produced by ligating antigenbinding region sequences from a (usually murine) monoclonal antibody to constant region sequences from a different species and/or isotype The first transgenic mice harboring immunoglobulin genes were made in the mid-1980s.[8] Though the majority of effort and funding in this field is currently focused toward human therapeutics, veterinary use of monoclonal antibodies also shows significant promise as a developing application Whereas several therapeutic monoclonal antibodies have been approved for use by the U.S Food and Drug Administration, none as yet has been approved where a transgenic animal was used as a production vehicle Using antibody production technologies in transgenic bioreactor systems, these products target a wide range of clinical ailments and are mostly in the preclinical stage of development EXPRESSION OF RECOMBINANT PROTEINS IN MEDIA OTHER THAN MILK Secretion of transgene-encoded proteins in the urine of transgenic animals was demonstrated using recombinant genes under the control of kidney-[9] or bladder-[10] specific regulatory sequences Expression of transgenes in the kidney or bladder of transgenic animals and subsequent secretion in the urine may provide some advantages over the mammary gland as a bioreactor, as the purification of proteins from urine may be facilitated by lower lipid and protein levels in comparison to milk Additionally, such animals can be used for production of recombinant proteins over the course of their entire life span RECOMBINANT PROTEIN PRODUCTION: HEALTH AND SAFETY ISSUES In addition to being structurally and functionally analogous to the natural plasma-derived protein, purified recombinant proteins must be free of pathogenic organisms Viral and bacterial contamination of human biopharmaceutical products produced in the blood or milk can be minimized by focusing prevention/eradication efforts on at least three levels of production: the transgenic animal donor, the medium in which the recombinant protein is produced, and the final product.[4] An initial key to minimizing the risk of contamination is to derive the transgenic donor animals from a source herd that is free from as many pathogens as possible Maintenance of these animals in a closed facility, the implementation of strict 845 monitoring procedures for various pathogens, and the use of animal husbandry practices that follow generally accepted practices (GAPs) and standard operating procedures should greatly reduce the entry of pathogens Though quite costly, one can develop pathogen-free herds of transgenic livestock Such a feat was recently achieved by introducing hysterotomy-derived transgenic piglets into an elaborate SPF barrier facility.[11] Diagnostic testing of this herd over the past years in this facility had revealed the absence of 35 major and minor swine pathogens including PRRS, parvovirus, leptospira, parainfluenza, and Streptococcus suis CONCLUSION While transgenic animal technology continues to open new and unexplored agricultural frontiers, molecular pharming efforts raise questions concerning regulatory and commercialization issues Although significant advances have been made since the inception of various clinical trials, the resources required to move the projects forward and the attendant financial risks have led a number of companies to curtail product development Various societal issues exist and will continue to influence the development of value-added animal products produced through transgenesis until transgenic products and foodstuffs are proven safe for human use and are accepted by a wide cross section of society ARTICLES OF FURTHER INTEREST Biotechnology: Stem Cell and Germ Cell Technology, p 146 Biotechnology: Transgenic Animals, p 149 Contributions to Society: Biomedical Research Models, p 239 Genetics: Molecular, p 466 Molecular Biology: Animal, p 653 Overall Contributions of Domestic Animals to Society, p 696 Phytases, p 704 Proteins, p 757 Transgenic Animals: Improved Performance, p 837 REFERENCES Houdebine, L M Production of pharmaceutical proteins from transgenic animals J Biotechnol 1994, 34 (3), 269 287 Young, M.W.; Okita, W.B.; Brown, M.; Curling, J.M 846 Production of biopharmaceutical proteins in the milk of transgenic dairy animals BioPharm 1997, 10 (6), 34 38 Pinkert, C.A The history and theory of transgenic animals Lab Anim 1997, 26 (8), 29 34 Clark, A.J.; Simons, P.; Wilmut, I.; Lathe, R Pharmaceut icals from transgenic livestock Trends Biotechnol 1987, (1), 20 24 Palmiter, R.D.; Brinster, R.L.; Hammer, R.E.; Trumbauer, M.E.; Rosenfeld, M.G.; Birnberg, N.C.; Evans, R.M Dramatic growth of mice that develop from eggs micro injected with metallothionein growth hormone fusion genes Nature 1982, 300 (5893), 611 615 Martin, M.J.; Pinkert, C.A Production of Transgenic Swine by DNA Microinjection In Transgenic Animal Technology: A Laboratory Handbook, 2nd Ed.; Pinkert, C.A., Ed.; Academic Press: San Diego, 2002; 307 336 Simons, J.P.; McClenaghan, M.; Clark, A.J Alteration of the quality of milk by expression of sheep b lactoglob Transgenic Animals: Secreted Products 10 11 ulin in transgenic mice Nature 1987, 328 (6130), 530 532 Storb, U.; Pinkert, C.; Arp, B.; Engler, P.; Gollahon, K.; Manz, J.; Brady, W.; Brinster, R.L Transgenic mice with mu and kappa genes encoding antiphosphorylcholine antibodies J Exp Med 1986, 64 (2), 627 641 Zbikowska, H.M.; Soukhareva, N.; Behnam, R.; Chang, R.; Drews, R.; Lubon, H.; Hammond, D.; Soukharev, S The use of the uromodulin promoter to target production of recombinant proteins into urine of transgenic animals Transgenic Res 2002, 11 (4), 425 435 Kerr, D.E.; Liang, F.; Bondioli, K.R.; Zhao, H.; Kreibich, G.; Wall, R.J.; Sun, T.T The bladder as a bioreactor: Urothelium production and secretion of growth hormone into urine Nat Biotechnol 1998, 16 (1), 75 79 Risdahl, J.; Edgerton, S.; Adams, C.; Martin, M.; Wiseman, B Establishing a Designated Pathogen Free Swine Col ony for Xenotransplantation, Proc 17th International Pig Veterinary Society Congress (IPVS), Ames, IA, June 4, 2002 Turkeys: Behavior, Management, and Well-Being C M Sherwin University of Bristol, Langford, U.K INTRODUCTION Each year, many millions of turkeys are reared for eating Methods of housing and managing these birds are diverse Turkeys are intelligent, inquisitive, social animals, and commercial rearing often conflicts with their psychological and physiological needs As a consequence, there are many welfare issues associated with turkey rearing, highlighted by comparing the domestic turkey with its ancestral species, the wild turkey of North America THE DOMESTIC TURKEY Modern domestic turkeys have been selected primarily for large body size and rapid growth rate Commercially, they are usually grown until they reach sexual maturity For males, this is approximately 20 weeks of age, when they can weigh over 20 kg, compared to a 3-year-old male wild turkey that weighs a mere kg The large body weight means that domestic turkeys are unable to fly, in contrast to their wild counterparts, and natural mating is replaced by artificial insemination to prevent injury to females Most turkeys derive from a small number of strains with homogenous white plumage, although some have retained the mottled appearance of the wild turkey Young domestic turkeys enthusiastically fly short distances, perch, and roost These behaviors become less prevalent with maturation, but adults readily climb onto objects such as straw bales Young birds perform spontaneous, frivolous running (frolicking), which has all the appearance of play Turkeys perform a wide diversity of behaviors, including comfort behaviors such as wing-flapping, feather-ruffling, leg-stretching, and dust-bathing They are highly social and become very distressed when isolated Many turkey behaviors are socially facilitated, i.e., expression of a behavior by one animal increases the tendency for this behavior to be performed by others Adults can recognize strangers[1] and placing any unfamiliar turkey into an established group will almost certainly result in that individual being attacked, sometimes fatally Turkeys are highly vocal, and social tension within the group can be monitored by Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019827 Copyright D 2005 by Marcel Dekker, Inc All rights reserved the birds’ vocalizations A high-pitched trill indicates the birds are becoming aggressive This can develop into intense sparring, where opponents leap at each other with their large, sharp talons and try to peck or grasp the other’s head Aggression increases in frequency and severity as the birds mature Maturing males spend a considerable proportion of their time sexually displaying This involves fanning the tail, drooping the wings, and erecting all body feathers including the beard (a tuft of black, modified hairlike feathers on the breast) The skin of the head, neck, and caruncles becomes bright blue and red, and the snood (an erectile appendage on the forehead) elongates The birds sneeze at regular intervals, followed by a rapid vibration of their tail Throughout, the birds strut slowly about with the neck arched backward and the breast thrust forward, emitting their characteristic gobbling call COMMERCIAL REARING Methods for rearing turkeys vary widely among producers and countries The following is typical for the United Kingdom Between and days of age, chicks are placed into small (2.5 m), circular brooding pens to ensure they encounter food and water To encourage feeding, they are kept under constant light for the first 48 h, and food is made widely accessible by scattering it on sheets of paper and in feeders After several days, the pens are removed, allowing the birds access to the entire rearing shed, which may contain tens of thousands of birds The birds remain here for several weeks, after which they are transported to another unit To assist thermoregulation, air temperature is maintained at 35°C for the first days, then lowered by approximately 3°C every days to 18°C at 37 days of age, and infrared heaters are usually provided for the first few days The vast majority of turkeys are reared indoors in purpose-built or modified buildings, of which there are two basic types The first type has slatted walls to allow ventilation (pole-barns) The second type has solid walls and no windows to allow lighting manipulations to optimize production (see the subsequent discussion) 847 848 The buildings are often very large, containing tens of thousands of birds as a single flock The substrate is usually deep litter, e.g., wood shavings, which relies on the controlled buildup of microbial flora, requiring skillful management Levels of CO2 and ammonia should not exceed 5000 ppm and ppm, respectively, and relative humidity should be maintained at 50 70% Ambient temperatures for adult turkeys are usually maintained at 18 21°C High temperatures should be avoided because the high metabolic rate of turkeys (up to 69 W/bird) makes them susceptible to heat stress, exacerbated by high stocking densities Handling during warm conditions should be avoided A variety of lighting schedules are used, e.g., continuous, intermittent, or long (23 h) photoperiods, to encourage feeding and accelerate growth.[2] Light intensity is usually low (e.g., < lux) to reduce feather-pecking (see the subsequent discussion) WELFARE ISSUES Intensive turkey production does not account for many of the birds’ psychological and physiological needs, resulting in welfare concerns Turkey chicks are precocial and are sustained by yolk reserves until days of age Learning to eat and drink appropriately during this time is essential, and it is common for a proportion of chicks to die (starve-out) from failing to learn, hence the use of brooding pens Chicks’ attention can be directed by tapping on the feeders and drinkers, thus simulating the behavior of the absent mother hen Space allowance for turkeys is often severely limited For example, a maximum permissible stocking density of 59.1 kg/m2 has been suggested.[3] This approximates to three adult 20-kg birds having to share m2, despite turkeys of this weight each requiring 1700 cm2 simply to stand without touching another bird.[4] The problems of small space allowance are exacerbated by the major influence of social facilitation if turkeys are to feed, drink, dust-bathe, etc simultaneously, then resources and space must be available in large quantities to avoid causing frustration The lighting manipulations used to optimize production can compromise welfare Long photoperiods combined with low light intensity can result in blindness from buphthalmia (distortions of the eye morphology) or retinal detachment, and can also result in distortion of the behavioral time budget Short photoperiods (8 h) can retard sexual development in males, and will also cause turkeys to eat in total darkness, possibly indicating an abnormally high motivation to feed resulting from selection for production characteristics A photoperiod of 12 16 h is Turkeys: Behavior, Management, and Well-Being adequate for turkeys to consume their daily feed requirement without any obvious adverse physiological or behavioral consequences Behavioral studies have shown that turkeys prefer light intensities higher than usually provided under commercial conditions In addition, low intensities make it difficult for humans to adequately inspect the birds Feather-pecking occurs frequently among turkeys and can begin at day of age This behavior is thought to be redirected foraging behavior, caused by providing birds with an impoverished foraging environment To reduce feather-pecking, turkeys are often beak-trimmed, which causes acute and possibly chronic pain Feather-pecking can be considerably reduced, at least in small groups (e.g., 100 birds), by providing supplementary ultraviolet radiation (turkeys are visually sensitive to UV; humans are not), pecking substrates (e.g., straw), and visual barriers to reduce social transmission of this behavior.[5] Other pecking substrates include chains, twine, vegetable matter, or food scattered in the substrate UV-reflective markings appear on young birds at the same time as feather-pecking becomes targeted toward these areas.[6] Turkeys also perform head-pecking, which becomes more frequent as they sexually mature When this occurs in small enclosures with few escape opportunities, the outcome is often rapidly fatal; healthy birds can be killed within hours Frequent monitoring is therefore essential, particularly of males approaching maturity Head injuries receive considerable attention from other birds, and headpecking often occurs after a relatively minor injury has been received during a fight or when lying down Birds with fresh injuries larger than cm should be closely monitored, and separation should be considered Individuals being reintroduced after separation are often immediately reattacked it might be impossible to reintroduce head-pecked individuals Fatal head-pecking can occur even in small (10 birds), stable groups Turkeys are normally reared in single-sex flocks If a male is inadvertently placed in a female flock, he may be aggressively victimized (henpecked) Females in male groups will be repeatedly mated, during which it is highly likely she will be injured from being trampled upon As with broiler fowl, turkeys often become less agile and experience walking difficulties as they become older This is due to a variety of diseases, anatomical changes from intensive selection for production traits, and poor husbandry Sometimes, the difficulty in locomotion can become so severe that birds refuse to walk and will die of starvation or thirst unless intervention occurs Locomotor problems cause the birds to spend long periods sitting on the substrate, which can lead to breast blisters and hock burns from high nitrogen content in the litter These have both welfare and economic consequences Poor litter Turkeys: Behavior, Management, and Well-Being quality can also cause foot-pad dermatitis, which can affect 98% of the flock Turkeys are prone to cardiovascular problems, so any physical exertion for them can be quite traumatic and may result in sudden death Domestic turkeys should be fed commercially available diets that have been developed to meet their nutritional requirements, although they will also benefit from fresh food as dietary enrichment Nutrient content, food quality, and feeding regimes must be carefully controlled to prevent leg abnormalities and other health and welfare problems associated with rapid growth rates CONCLUSION Despite years of intensive artificial selection, which have considerably changed aspects of their morphology and behavior compared to the wild turkey, the domestic turkey is an intelligent, social animal that displays a wide range of behavior and has both psychological and physiological needs Intensive commercial rearing conflicts with many of these needs and as a consequence, there are many compromises of welfare Future research should include alleviation of these compromises as a priority 849 ACKNOWLEDGMENT C M Sherwin received the UFAW Hume Research Fellowship during preparation of this article REFERENCES Buchwalder, T.; Huber Eicher, B A brief report on ag gressive interactions within and between groups of domestic turkeys (Meleagris gallopavo) Appl Anim Behav Sci 2003, 84, 75 80 Nixey, C Lighting for the production and welfare of turkeys World’s Poultry Sci J 1994, 50, 292 294 Farm Animal Welfare Council Report on the Welfare of Turkeys; Tolworth: UK, 1995; 13 15 Ellerbrock, S.; Knierim, U Static space requirements of male meat turkeys Vet Rec 2002, 151, 54 57 Sherwin, C.M.; Lewis, P.D.; Perry, G.C Effects of environmental enrichment, fluorescent and intermittent lighting on injurious pecking amongst male turkey poults Br Poultry Sci 1999, 40, 592 598 Sherwin, C.M.; Devereux, C.L A preliminary investigation of ultraviolet visible markings on domestic turkey chicks and a possible role in injurious pecking Br Poultry Sci 1999, 40, 429 433 Turkeys: Nutrition Management Todd J Applegate Purdue University, West Lafayette, Indiana, U.S.A INTRODUCTION Compounds That Potentiate Early Growth In 1970, a male turkey averaged only 16.9 pounds and required 3.10 pounds of feed for every pound of gain at 18 weeks of age Today, genetic and nutritional improvements have increased growth such that the average male turkey at 18 weeks of age weighs 33.4 pounds and requires only 2.52 pounds of feed for every pound of gain (Fig 1).[1] Because average body weights for hens are only 21.75 pounds, the industry has developed separate markets and rearing practices for male and female turkeys Turkey toms (males) are reared primarily for cut-out and further processed products, whereas hens are reared for whole-bird and parts markets This phenomenal increase in growth has not come without its share of health, metabolic, structural, and nutritional issues Primary issues in the area of nutrition for modern turkey production include 1) transition diets at the start of life; 2) feeding to maximize gastrointestinal (GIT) health; 3) bone integrity; 4) minimizing environmental impact; and 5) maximizing muscle mass and meat quality A practical approach to applying compounds to stimulate early growth has been investigated by researchers at North Carolina State University by studying the effects of in ovo administration of peptide YY In other species, peptide YY has demonstrated effects of inhibiting gut motility and stimulating small intestinal absorption of glucose Application of peptide YY in ovo at transfer improves body weight up to days of age in the poult Administration of nutrients into the amnion of the egg prior to when the poult imbibes the remaining amniotic fluid before hatching has positive effects on intestinal maturation and poult growth after hatch FROM THE HATCHERY TO THE FARM—MAXIMIZING EARLY GROWTH Early access to feed and water after hatching is important to ensure that young poults have a good start and are able to realize their growth potential Often, turkeys will hatch over a hatching window of 48 hours or more It is not uncommon, therefore that a proportion of the poults placed (given access to food and water) on the farm have hatched nearly 48 to 72 hours prior to placement Early hatching, hatchery services, and transportation to the farm contribute to the challenge of delivering nutrients to the poult soon after exiting the egg During the first week after hatching, the poult’s small intestine increases in weight ninefold and doubles in length.[2] Delayed access to feed greatly affects intestinal morphology and growth of the bird after feeding up to four weeks of age Part of this delay can be attributed to damage to microvilli and crypt cell structure in the small intestine, which can be adversely affected up to nine days after hatching 850 Starter Diet Composition Diet composition has a profound effect on how the poult makes the transition to its new metabolic state.[3] Traditional perception by the industry is that fat supplementation should be minimized for starting hatchlings From a digestibility standpoint, research with feeding of animal fats and animal/vegetable fat blends demonstrates that young hatchlings not digest saturated fatty acids efficiently However, unsaturated fatty acids are highly digestible (80 to 85%) and may actually ease the metabolic shift after hatching Caution should be used, however, as unsaturated fats typically are easily oxidized, rendering the fat rancid Others may contend that a high proportion of energy from carbohydrate is needed to facilitate a shift in metabolism (from deriving energy from yolk lipid to assimilation of carbohydrate from an external diet) However, when diets containing a high proportion of energy from corn (carbohydrate) are fed, researchers from Ohio State University noted that 30 to 50% of poults fed the carbohydrate-based diet have plasma glucose concentrations above 500 mg/dL days after feeding, which is more than twice the normal concentration Because the young poult has a very high crude protein requirement (28%), nutritionists may have a tendency to include much soybean meal in starting diets However, soybean meal contains a high proportion of nonstarch polysaccharides and is very poorly digested Therefore, Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019831 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Turkeys: Nutrition Management 851 Fig Average live body weight of turkey toms (18 weeks) and hens (14 weeks) (From Ref [1].) prestarter or starter diets containing a high proportion of soybean meal may not provide the required amount of calculated energy and may suppress early growth In the case of the turkey poult, the initial diet should contain less than 40% of the diet from soybean meal Typically, formulation will include approximately to 10% fish and/ or meat and bone meal Several companies are currently marketing specialized diets to be used as the first diet at placement, or in transport boxes before arrival at the farm These specialized diets are formulated with more easily digested nutrients, such as egg albumin as a protein source, and may also contain mixtures of bacteria (termed competitive exclusion products) to establish a normal microflora and exclude possible pathogens NUTRIENT IMPACT ON MUSCLE GROWTH AND QUALITY Breast meat has become the primary retail product for commercial turkey production, so much nutritional research has focused on maximizing muscle growth and meat quality Proteins and their component amino acids are one of the primary cost components of turkey diets, but also one of the primary drivers of muscle growth The turkey industry typically formulates diets based on a crude protein basis, which can supply amino acids in excess of what the animal can either digest or metabolize As research in this area progresses, however, knowledge of the amino acid digestibility of typical feed ingredients and formulating with an ideal protein ratio (theoretical exact balance of digested amino acids for metabolic needs) will allow greater precision in formulation.[4] This added precision should considerably reduce the amount of nitrogen that is excreted by turkeys Additionally, recent nutritional advances have allowed the turkey industry to greatly improve the functional characteristics of turkey meat For example, supplementation with vitamin E well above nutrient requirements for prevention of classical deficiency symptoms reduces lipid oxidation and improves the shelf life of both raw and ground turkey meat Furthermore, vitamin E supplementation at higher concentrations in the diet reduces the incidence of pale, soft, and exudative (PSE) meat often encountered in heat-stressed birds.[5] Much of this effect can be related to alterations in glycolytic characteristics during the muscle rigor process Other feed additives, such as betaine, show promise in aiding osmoregulation during coccidial challenges as well as providing a higher water-soluble methyl donor source, which has demonstrated effects on muscle growth in turkeys NUTRITION AND BONE INTEGRITY Genetic improvements in the growth potential of commercial turkeys have not readily translated into proportional carcass growth and body conformation For example, leg-associated disorders were a considerable issue for the industry during the late 1980s, until legshank width and conformation traits became selection criteria by breeding companies Today, bone integrity is still a considerable economic issue as companies begin to market larger birds (over 40 pounds) at older ages Nutritionally speaking, adequate dietary amino acid and 852 protein levels have demonstrated effects on muscle growth, and they are associated with pronounced effects on the lower skeletal axis as well.[6] In fast-growing strains, spiral fractures of the femur occur in small percentages of toms between 16 and 19 weeks of age Although it is unknown whether there is a nutritional cause, preliminary results suggest a problem in collagen fibril distribution/arrangement, along with areas of lower calcification Other skeletal issues, such as osteomyelitis complex, are of considerable economic impact to the industry, because carcasses displaying the lesion are condemned due to a high incidence of Staphylococcus aureus Supplementation with either 1,25 dihydroxy vitamin D3 or 25-hydroxy vitamin D3 improves skeletal integrity and reduces the incidence of osteomyelitis complex during an immune/bacterial challenge.[7] Turkeys: Nutrition Management CONCLUSION Nutritional issues facing the turkey industry at present include 1) transition diets at the start of life; 2) feeding to maximize GIT health; 3) maintaining bone integrity; 4) minimizing environmental impact; and 5) maximizing muscle mass and meat quality Unfortunately, nutritional research for turkeys has not kept pace with substantial gains in performance Fundamental research addressing bird requirements, feedstuff utilization, maintaining performance and GIT health without antibiotics, and minimizing environmental impact will be critical for the continued success of turkey production REFERENCES NUTRITION AND THE ENVIRONMENT Phosphorus (P) has emerged as an environmental issue with respect to surface-water quality In order for animal agriculture to comply with new P-based land application regulations and the reductions in watershed scale nutrient loading imposed by total maximum daily load (TMDL) agreements in many U.S states, it is imperative that 1) excreta P produced by the animals be minimized as much as possible through efficient animal nutrient management practices, such as dietary modification; and 2) the effects of dietary changes on the forms, availability, and transport of P in manure-amended soils be evaluated Recent studies at Michigan State University and Purdue University demonstrate that the NRC nutrient recommendations[8] for P for turkeys are adequate for maximizing growth Therefore, industry diets, containing at least 30% greater P, were not justified and led to substantially greater P excretion Further reductions in litter P excretion can be achieved when microbial phytase (after reducing dietary P by 0.08%) or 25hydroxy vitamin D3 (after reducing dietary P by 0.03%) are fed.[9] Ferket, P.R Growth of toms improves substantially WATT Poult USA July 2003, 38 48 Sell, J.L Physiological limitations and potential for improvement in gastrointestinal tract function of poultry J Appl Poult Res 1996, 5, 96 101 Lilburn, M.S Ingredient quality and its impact on digestion and absorption in poultry J Appl Poult Res 1996, 5, 78 81 Firman, J.D.; Boling, S.D Ideal protein in turkeys Poult Sci 1998, 77, 105 110 Olivo, R.; Soares, A.L.; Ida, E.I.; Shimokomaki, M Dietary vitamin E inhibits poultry PSE and improves meat func tional properties J Food Biochem 2001, 25, 271 283 Turner, K.A.; Lilburn, M.S The effect of early protein restriction (zero to eight weeks) on skeletal development in turkey toms from two to eighteen weeks Poult Sci 1992, 71, 1680 1686 Huff, G.R.; Huff, W.E.; Balog, J.M.; Rath, N.C.; Xie, H.; Horst, R.L Effect of dietary supplementation with vitamin D metabolites in and experimental model of turkey osteomyelitis complex Poult Sci 2002, 81, 958 965 National Research Council Nutrient Requirements of Domestic Animals In Nutrient Requirements of Poultry, 9th Revised Edition; National Academy Press, 1994 Applegate, T.J.; Angel, R Does dietary phytase supple mentation increase phosphorus solubility in poultry manure? Proc Carolina Poult Nutr Conf 2003, 13, 88 104  ... being completely resistant to infection These results clearly demonstrated the potential of a transgene to combat one of the most prevalent diseases of dairy cattle The same lysostaphin transgene... services, and transportation to the farm contribute to the challenge of delivering nutrients to the poult soon after exiting the egg During the first week after hatching, the poult’s small intestine increases... at North Carolina State University by studying the effects of in ovo administration of peptide YY In other species, peptide YY has demonstrated effects of inhibiting gut motility and stimulating