Encyclopedia Of Animal Science - P ppsx

Pathogens in Animal Products: Major Biological Hazards John N Sofos Colorado State University, Fort Collins, Colorado, U.S.A INTRODUCTION The most severe biological hazards in foods are pathogenic bacteria, which may cause illness as direct agents (infection) or through production of various toxins (intoxication), while additional biological hazards include parasitic and viral agents Typical clinical symptoms of foodborne bacterial and viral diseases include acute diarrhea, abdominal cramps, vomiting, or some other manifestation in the gastrointestinal tract In addition, syndromes associated with the central nervous system or various organs, as well as various chronic sequelae, may also be the direct or indirect result of foodborne pathogenic bacteria Individuals with suppressed or compromised immune systems are more susceptible to severe foodborne microbial illness Prions found in animal central nervous tissue are considered a potential newer type of hazard, leading to development of transmissible spongiform encephalopathies (TSE) According to the U.S Centers for Disease Control and Prevention (CDC), it is estimated that for the period 1993 1997, meat and poultry were responsible for 17.3% of the total outbreaks of known vehicle of transmission and for 11.4% of the corresponding cases (www.cdc.gov) BACTERIAL PATHOGENS Escherichia coli O157:H7 E coli are mostly harmless colonizers of the gastrointestinal tract of warm-blooded animals, including humans; certain strains, however, cause diarrheal illness.[1] Some strains produce Shigalike toxins (SLT) or verotoxins (VT) and are classified as enterohemorrhagic E coli (EHEC) or Shigalike toxin producing (STEC), or as verocytotoxigenic (VTEC), with E coli O157:H7 being the predominant EHEC serotype E coli O157:H7 are Gram-negative, facultatively anaerobic, non-spore-forming rods that are mostly motile They can grow at temperatures of 46°C (optimum 35 40°C), in water activities of ! 0.95, and at pH values of 4.4 9.0 (optimum 6.0 7.0).[1] E coli 698 O157:H7 usually cause illness through fecal oral transmission or through consumption of contaminated foods, with the majority of outbreaks involving consumption of undercooked ground beef Other foods, such as fruit juices, cantaloupe, and seed sprouts for salads, have also been associated with illness through fecal cross-contamination Ingestion of cells (! 10 cells) is followed (3 days incubation) by mild or severe bloody diarrhea (hemorrhagic colitis) and hemolytic uremic syndrome (HUS).[1] E coli O157:H7 are declared an adulterant for raw ground beef and other nonintact beef products by the U.S Department of Agriculture Food Safety and Inspection Service (USDA/FSIS), and are estimated to cause a total of 73,000 cases of illness in the United States each year.[2] Listeria monocytogenes Listeria are non-spore-forming, aerobic, microaerophilic or facultatively anaerobic, Gram-positive rods that are motile by means of peritrichous flagella.[1] The organism is ubiquitous in the environment and may be harbored in many animals Its presence in plant floors, walls, drains, condensed and standing water, and food residues on processing equipment, and its involvement in certain highly fatal (20 30%) foodborne outbreaks, make this a pathogen of major concern As a psychrotroph, L monocytogenes can grow at À to 45°C (optimum 30 37°C) Growth occurs at pH 4.4 9.4 and in water activities above 0.92 Foods of concern for transmission of listeriosis include non-shelf-stable, ready-to-eat meat and poultry products, deli-type foods, soft cheeses, seafood, and unpasteurized dairy products Although there is an enteric form of listeriosis, the major concern is associated with the nonenteric infection, which affects mainly the central nervous system (meningitis, meningoencephalitis, bacteremia), and may result in stillbirth, fetal death, or spontaneous abortion in pregnant women The infectious dose is believed to be ! 100 cells/g of food, and the incubation period a few days to months.[1] The pathogen is estimated to be responsible for approximately 0.02% of the cases of foodborne illness in the United States and 28% of the deaths.[2] Encyclopedia of Animal Science DOI: 10.1081/E EAS 120030467 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Pathogens in Animal Products: Major Biological Hazards 699 Salmonella Other Bacterial Pathogens This long-known pathogen is a Gram-negative, facultatively anaerobic, non-spore-forming rod; its only two species are Salmonella enterica, possessing six subspecies, and Salmonella bongori There are approximately 2600 Salmonella serotypes, of which Salmonella Typhimurium and Salmonella Enteritidis are the most prevalent in the United States.[2]Salmonella can grow at temperatures of 5.2 46.2°C, pH values of 3.8 9.5, and in water activity ! 0.93 The primary reservoir for Salmonella is the gastrointestinal tract of infected animal hosts or carriers, which serve as sources of contamination for foods and the environment Nontyphoidal Salmonella strains usually cause gastroenteritis after an incubation period of hours to days, resulting in diarrhea, nausea, mild fever, chills, vomiting, and abdominal cramping Infectious doses of Salmonella (10 10,000 cells) depend on serotype, vehicle of transmission, and on the individual’s susceptibility.[1] A variety of food products, including meat, poultry, and dairy products, have been implicated in the transmission of salmonellosis Salmonella causes millions of illnesses and approximately 30% of the total estimated deaths from foodborne disease in the United Sates annually.[2] Several other bacterial pathogens are associated with food products of animal origin, but their contribution to foodborne disease has been overshadowed by the impact of the aforementioned four pathogens in recent years They include additional pathogenic serotypes of E coli, Yersinia enterocolitica (a psychrotroph of enteric origin), Staphylococcus aureus (a Gram-positive, heat-stable, enterotoxin-producing mesophile), Clostridium botulinum (a deadly, neurotoxin-producing spore-former), Clostridium perfringens (a common cause of gastrointestinal discomfort of short duration), and Bacillus cereus (a sporeforming mesophile or psychrotroph causing diarrheal or emetic illness) Detailed information on these and other pathogens of less current concern can be found in several publications[1–8] and at www.cfsan.fda.gov Campylobacter This organism consists of Gram-negative non-sporeforming, slender and curved rods, which, along with the single, polar flagellum located at one or both ends of the cell, cause its characteristic corkscrew-type motility.[1] As microaerophilic, Campylobacter grows best in environments of 2.0 5.0% oxygen and 5.0 10.0% carbon dioxide, while growth is inhibited at normal oxygen levels It grows at 30 45°C (optimum 37 42°C), pH values of 4.9 8.0 (optimum 6.5 7.5), and in water activities above 0.91 Campylobacteriosis may result from as few as 500 cells (2 10 days incubation), and the low-fatality infection typically involves acute colitis combined with fever, malaise, abdominal pain, headache, watery or sticky diarrhea with minor traces of blood (occult), inflammation of the lamina propria, and crypt abscesses Infection may lead to additional sequelae, including an acute paralytic disease of the peripheral nervous system known as Guillain Barre syndrome and an autoimmune disease known as Reiter’s syndrome.[1] Campylobacteriosis outbreaks have involved consumption of milk, water, and foods exposed to fecal contamination or cross-contamination.[1] Although poultry meat is considered a major source of Campylobacter, it is believed that a large portion of its millions of cases of foodborne illness occurs through cross-contamination OTHER BIOLOGICAL HAZARDS Food-producing animals may also serve as sources of parasitic and viral disease agents.[1] For example, swine may be involved in the transmission of trichinosis (Trichinella spiralis), sarcocystosis (Sarcocystis spp.), and toxoplasmosis (Toxoplasma gondii); and poultry of toxoplasmosis, whereas beef cattle may transmit tapeworms (Taenia spp.) and Sarcocystis spp or serve as indirect vectors for the transmission of Cryptosporidium parvum (cryptosporidiosis) through water contaminated with feces.[9] Viral agents, such as Norovirus, hepatitis A, and enteroviruses, are responsible for most foodborne disease cases in the United States,[2] but their transmission is mostly associated with poor sanitation, cross-contamination during preparation and serving, or inadequate cooking Bovine spongiform encephalopathy (BSE) has emerged as a major animal health issue in recent years, especially because of its potential involvement in human transmissible spongiform encephalopathies (TSE) such as a new variant, Creutzfeldt Jakob Disease (vCJD) Evidence indicates that BSE is caused by prions found in central nervous tissue, and originated in cattle fed ruminant byproducts In the 1990s, the United States established a number of measures to prevent the emergence of this problem in this country They included a ban on importation of live ruminants and their products from countries with native BSE, immunohistochemical examinations of brains of cattle condemned for nervous system disorders, and a ban on the use of ruminant materials in meat and bone meal feeds for ruminants.[9] However, in December 2003, the first cow with BSE was detected in the United States, following another single case in Canada earlier that year Following this event, additional measures were announced by the USDA/FSIS and the Food and 700 Drug Administration (FDA) in efforts to prevent spread of the problem and to better protect public health These measures included banning use of downer (unable to stand and walk) cattle from human food, holding carcasses of cattle tested for BSE until results are confirmed, prohibiting stunning of cattle with air-injection guns, banning from the food supply specified risk materials (brain, skull, eyes, spinal cord, small intestines, etc.) of cattle over 30 months of age and the small intestine of cattle of all ages, increasing process controls for material obtained with advanced meat recovery systems, banning use of mechanically separated meat in food products, and banning from FDA-regulated foods, dietary supplements, and cosmetics use of the previous materials CONCLUSION Food products of animal origin may be contaminated with biological hazards, including pathogens to human health Such hazards include bacterial, viral, and parasitic agents, and prions associated with transmissible spongiform encephalopathies Health problems associated with these hazards range from short-term, mild forms of gastrointestinal discomfort, to severe damage of various tissues and organs, death, chronic sequelae, or long-term medical syndromes ARTICLES OF FURTHER INTEREST Animal Source Food: Quality and Safety Meat and Poultry, p 33 Animal Source Food: Quality and Safety Milk and Eggs, p 36 Antibiotics: Microbial Resistance, p 39 Eggs: Marketing, p 311 Eggs: Processing, Inspection, and Grading, p 317 Future of Animal Agriculture: Demand for Animal Products, p 432 Pathogens in Animal Products: Major Biological Hazards Lower Digestive Tract Microbiology, p 585 Molecular Biology: Microbial, p 657 Pathogens in Animal Products: Sources and Control, p 701 Poultry Meat: Inspection/Grading, p 735 Probiotics, p 754 Rumen Microbiology, p 773 Sheep Milk and Milk Production: Processing and Marketing, p 794 REFERENCES Bacon, R.T.; Sofos, J.N Characteristics of Biological Hazards In Food Safety Handbook; Schmidt, R.H., Rodrick, G.E., Eds.; Wiley Interscience: Hoboken, NJ, USA, 2003; 157 195 Mead, P.S.; Slutsker, L.; Dietz, V.; McCaig, L.F.; Bresee, J.S.; Shapiro, C.; Griffin, P.M.; Tauxe, R.V Food related illness and death in the United States Emerg Infect Dis 1999, 5, 607 625 Doyle, M.P Foodborne Bacterial Pathogens; Marcel Dekker, Inc.: New York, NY, USA, 1989 Cliver, D.O.; Riemann, H.P Foodborne Diseases, 2nd Ed.; Academic Press: San Diego, CA, USA, 2002 Blackburn, C.W.; McClure, P.J Foodborne Pathogens, Hazards, Risk Analysis and Control; CRC Press/Woodhead Publishing Limited: Cambridge, UK, 2002 Labbe, R.G.; Garcia, S Guide to Foodborne Pathogens; Wiley Interscience: New York, NY, USA, 2001 Hui, Y.H; Pierson, M.D.; Gorham, J.R Foodborne Diseases Handbook, 2nd Ed.; Bacterial Pathogens, Marcel Dekker, Inc.: New York, NY, USA, 2001; Vol Hui, Y.H; Sattar, S.A.; Murrell, K.D.; Nip, W K.; Stanfield, P.S Foodborne Diseases Handbook, 2nd Ed.; Viruses, Parasites, Pathogens, and HACCP, Marcel Dekker, Inc.: New York, NY, USA, 2001; Vol CAST (Council for Agricultural Science and Technology) Intervention Strategies for the Microbiological Safety of Foods of Animal Origin; Council for Agricultural Science and Technology: Ames, IA, USA, January 2004 Issue Paper #25 Pathogens in Animal Products: Sources and Control John N Sofos Colorado State University, Fort Collins, Colorado, U.S.A INTRODUCTION Food products of animal origin (i.e., fresh, processed, and ready-to-eat meat and poultry products, eggs, milk, and other dairy products) may be contaminated during harvesting, processing, and handling Because they are rich in nutrients, they support growth of various spoilage and pathogenic microorganisms if not properly handled and preserved Spoilage microorganisms damage product quality and lead to reduced food supplies and economic losses, whereas pathogens may cause mild, severe, brief, or chronic human illness, or death Knowledge of sources of contamination and of the properties of foodborne pathogens allows application of proper procedures for pathogen control and enhancement of food safety Sources and control of pathogens are addressed in this article, while major biological hazards are discussed elsewhere in this encyclopedia CONTAMINATION SOURCES Animal production and product processing and handling practices result in contamination with Gram-negative and Gram-positive bacteria, yeasts, molds, parasites, and viruses, but the presence of pathogens in animal products processed under sanitary and hygienic conditions should generally be infrequent and at low levels Contamination, however, is unpredictable Thus, any raw, unprocessed, uncooked food should be considered as potentially contaminated with pathogens In general, before slaughter, internal muscle tissues of healthy animals and birds can be considered sterile, whereas lymph nodes and certain organs (e.g., liver) may carry low levels of microbial contamination In contrast, animal surfaces exposed to the environment such as hides, pelts, feathers, fleece, the mouth, and the gastrointestinal tract may be heavily contaminated.[1–5] Contamination from soil, decaying matter, and animal waste is transmitted to water, air, pastures, and animal feeds, which may carry contamination naturally or may be cross-contaminated with manure Additional sources of biological hazards may include rodents, mice, birds, insects, and transportation vehicles or crates for animals, which may contribute to cross-contamination, although Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019744 Copyright D 2005 by Marcel Dekker, Inc All rights reserved the extent of this is unknown.[1–6] Pathogen prevalence may vary with animal type and age, geographic region, and season, and is usually higher during the warmer months Animal manure may contaminate water used for drinking or to irrigate or wash plant crops, resulting in cross-contamination of other foods The extent of microbial transfer from the aforementioned sources to food products of animal origin depends on sanitation and hygienic practices; product handling and processing procedures; and conditions of storage, distribution, retailing, preparation for consumption, and serving.[1–5] Animal parts and manure serve as sources of contamination of milk, shell eggs, meat and poultry carcasses and their products, and the environment, leading to cross-contamination of other foods Meat and poultry are contaminated during slaughtering, dressing, chilling, and cutting processes, when animals’ muscles are exposed to the environment Sources of meat contamination include air, water, feces, hides, intestines, lymph nodes, processing equipment, utensils, and humans During milking, milk is contaminated by the animal and its environment, as well as by milking equipment and utensils Eggs may be contaminated through shell penetration or during egg-breaking, whereas internally contaminated eggs may carry Salmonella serotype Enteritidis transmitted through transovarian infection of chickens.[1–3,6] The types and levels of microorganisms contaminating a product and subsequent product handling may have important consequences on product quality and safety CONTROL Concern about animal food products serving as vehicles of foodborne biological hazards has led to the establishment of regulatory requirements aimed at improving their hygienic state Recent developments have included the complete change of the U.S meat and poultry inspection system, which has been in place since the early 1900s The new U.S Meat and Poultry Inspection Regulation[7] requires federally inspected meat and poultry plants to implement the hazard analysis critical control point (HACCP) system for the management of process controls.[8] The application of process controls[6,9,10] for 701 702 enhancement of the microbiological condition and assurance of the safety of meat and poultry products involves three approaches: 1) control of sources and processes to decrease the likelihood of contamination during product harvesting; 2) implementation of procedures to decontaminate products such as carcasses during processing; and 3) application of technologies to inactivate or control pathogens in ready-to-eat products These approaches are applicable for pathogen control in all foods and aim at minimizing initial contamination, destroying contamination in some products, and inhibiting proliferation of contamination in others.[9–12] Approaches to controlling bacterial pathogens in live animals include use of feed additives, diet modification, antimicrobial treatments, competitive exclusion microorganisms, treatment with bacteriophages, administration of vaccines, improved husbandry practices, etc.[12] With the exception of application of good production practices, all these approaches are still in the experimental stage Decontamination interventions applied to meat-animal carcasses include animal cleaning; dehairing; spot-cleaning of carcasses before evisceration by knife-trimming; steaming and vacuuming; and spraying, rinsing, or deluging of carcasses before evisceration and/or before chilling with hot water, chemical solutions, or steam.[10] These processes only reduce contamination levels because it is difficult to eliminate microbial contamination and still maintain the raw state properties of foods Processes aimed at destruction of contamination during product processing include use of heat and, to a lesser extent, ionizing radiation or high-pressure processing Approaches aiming to inhibit microbial growth are based on low storage temperatures, drying (evaporation, concentration), binding of water levels (salting, sugaring) available for microbial growth (water activity), addition of acids (low pH), fermentation (low pH, production of antimicrobials), packaging under such modified atmospheres as vacuum, and use of chemical preservatives.[1–3,9] Parasites may be inactivated by proper cooking, freezing, irradiation, salting, or application of chemicals Viruses can be controlled by application of proper cooking, sanitation, and hygienic procedures Interventions available to minimize pathogen contamination of milk include health management programs for dairy herds; employment of hygienic practices during milking, storing, and distribution of milk; and pasteurization.[6] It should be noted that pathogen-free raw milk cannot be ensured, and consumers should be advised not to drink raw milk or consume dairy products made with raw milk Microbiological concerns associated with processed dairy products include potential survival of pathogens such as Salmonella in cheeses and growth of Listeria monocytogenes in certain soft cheeses Whereas outbreaks of salmonellosis are rarely associated with Pathogens in Animal Products: Sources and Control natural cheeses, listeriosis has been associated with consumption of fluid milk and soft cheeses when growth occurred before consumption These concerns may be addressed through prevention of contamination after pasteurization of milk and during the manufacture of cheese products, and through control of the environment to prevent contamination.[6] Presence of Salmonella Enteritidis in shell eggs is an important concern because it may be introduced through transovarian infection The pathogen can be destroyed by proper cooking, liquid egg pasteurization, or in shell egg pasteurization.[6] A decrease in Salmonella Enteritidis egg-associated illness in the United States may be the result of improved farm management practices, procedures for detecting and controlling Salmonella Enteritidis contaminated flocks, timely collection of eggs, storage of eggs at low temperatures, consumer education for safe egg handling, and pasteurization of eggs from infected flocks A significant decrease in Salmonella Enteritidis infections reported in the United Kingdom may be attributed to vaccination of the egg-laying flocks.[6] CONCLUSION Food products of animal origin are expected by nature to be contaminated with microorganisms, including some that are pathogenic to humans.[13] These pathogens may cause illness, ranging from mild gastrointestinal discomfort to severe acute or chronic illness or death Extent, prevalence, and type of contamination are influenced by sanitary, hygienic, and processing conditions during handling of the products at all stages of the food chain It is important to realize that control of pathogens and management of food safety risks should be based on an integrated approach that applies to all sectors from the producer to the processor, distributor, packer, retailer, food service worker, and consumer Interventions applied during processing include sanitation, decontamination, heating, chilling, freezing, drying, fermentation, use of chemicals as acidulants or antimicrobials, packaging, proper storage and distribution, and appropriate handling and preparation for consumption Proper application of control processes yields products that should be safe for consumption following proper cooking and serving Consumers should be advised to properly handle and prepare all foods, including those of animal origin, and to follow labeling instructions Foods should be stored and handled in conditions that minimize cross-contamination (i.e., in a clean and sanitary environment), properly cooked (e.g., ground beef cooked at 160°F), and stored or held at the correct temperatures (cold: under 40°F; hot: above 140°F), and for the indicated length of time Pathogens in Animal Products: Sources and Control ARTICLES OF FURTHER INTEREST Animal Source Food: Quality and Safety Meat and Poultry, p 33 Animal Source Food: Quality and Safety Milk and Eggs, p 36 Antibiotics: Microbial Resistance, p 39 Eggs: Marketing, p 311 Eggs: Processing, Inspection, and Grading, p 317 Future of Animal Agriculture: Demand for Animal Products, p 432 Lower Digestive Tract Microbiology, p 585 Molecular Biology: Microbial, p 657 Pathogens in Animal Products: Major Biological Hazards, p 698 Poultry Meat: Inspection/Grading, p 735 Probiotics, p 754 Rumen Microbiology, p 773 Sheep Milk and Milk Production: Processing and Marketing, p 794 703 10 REFERENCES Sofos, J.N Microbial Growth and Its Control in Meat Poultry and Fish In Quality Attributes and Their Measure ments in Meat, Poultry and Fish Products; Pearson, A.M., Dutson, T.R., Eds.; Blackie Academic and Professional: Glasgow, UK, 1994; 353 403 Koutsoumanis, K.P.; Sofos, J.N Microbial Contamination of Carcasses and Cuts In Encyclopedia of Meat Sciences; Elsevier: Oxford, UK, 2004 in press Koutsoumanis, K.P.; Geornaras, I.; Sofos, J.N Microbiol ogy of Land Muscle Foods In Handbook of Food Science; Hui, Y.H., Ed.; Marcel Dekker, Inc.: New York, NY, USA, 2004, in press 11 12 13 ICMSF (International Commission on Microbiological Specifications for Foods) Microorganisms in Foods 6, Microbial Ecology of Food Commodities; Blackie Aca demic and Professional: London, UK, 1998 Davies, A.; Board, R The Microbiology of Meat and Poultry; Blackie Academic and Professional: London, UK, 1998 CAST (Council for Agricultural Science and Technology) Intervention Strategies for the Microbiological Safety of Food of Animal Origin, Issue Paper; Council for Agricultural Science and Technology: Ames, IA, USA, January 2004 Issue Paper # 25 FSIS (Food Safety and Inspection Service) Pathogen reduction; hazard analysis and critical control point (HACCP) systems: Final rule Federal Register 1996, 61, 38805 38989 9CFR Part 304 NACMCF (National Advisory Committee on Microbio logical Criteria for Foods) Hazard analysis and critical control point principles and application guidelines J Food Prot 1998, 61, 762 775 Juneja, V.K.; Sofos, J.N Control of Foodborne Microor ganisms; Marcel Dekker, Inc.: New York, NY, USA, 2002 Sofos, J.N.; Smith, G.C Nonacid meat decontamination technologies: Model studies and commercial applications Int J Food Microbiol 1998, 44, 171 188 Samelis, J.; Sofos, J.N Strategies to Control Stress Adapted Pathogens In Microbial Stress Adaptation and Food Safety; Yousef, A.E., Juneja, V.K., Eds.; CRC Press: Boca Raton, FL, USA, 2003 Sofos, J.N Approaches to Pre Harvest Food Safety Assurance In Food Safety Assurance and Veterinary Public Health; Volume 1, Food Safety Assurance in the Pre Harvest Phase; Smulders, F.J.M., Collins, J.D., Eds.; Wageningen Academic Publishers: Wageningen, The Netherlands, 2002; 23 48 Sofos, J.N Pathogens in Animal Products: Major Biolog ical Hazards In Encyclopedia of Animal Science; Pond, W.G., Bell, A.W., Eds.; Marcel Dekker, Inc.: New York, 2004 Phytases Xin Gen Lei Cornell University, Ithaca, New York, U.S.A Jesus M Porres Universidad de Granada, Grenada, Spain INTRODUCTION Phytases are meso-inositol hexaphosphate phosphohydrolases that catalyze the initiation of the stepwise phosphate splitting of phytic acid or phytate to lower inositol phosphate esters and inorganic phosphate (Fig 1) These enzymes have emerged as effective tools to improve phosphorus nutrition and to protect the environment from phosphorus pollution in animal production Although phosphorus is an essential nutrient to all species, 60 80% of phosphorus in feeds of plant origin is in the form of phytate (myo-inositol hexakisphosphate) that is poorly available to simple-stomached animals such as swine, poultry, and the preruminant calves, due to the lack of phytases in their gastrointestinal tracts As a result, a large portion of feed phosphorus is not utilized by them and ends up in manure, causing environmental pollution Meanwhile, expensive and nonrenewable inorganic phosphorus needs to be added to diets for these species to meet their nutrient requirements for phosphorus GENES, PROTEINS, AND PROPERTIES A number of phytase genes and proteins have been identified from microorganisms and plants after the isolation of the very first phytase (PhyA) protein and DNA sequence from Aspergillus niger.[1] It remains unclear whether phytase is expressed in animal tissues PhyA and most fungal phytases have molecular mass ranging from 80 120 kDa, with 10 or so N-glycosylation sites in the approximately 1.4-kb DNA sequences.[1] The average molecular masses of most bacterial phytases range from 40 to 55 kDa.[2] Plant phytases isolated from corn, wheat, lupine, oat, or barley have molecular sizes ranging from 47 to 76 kDa.[3] Most identified phytases, but not all, belong to a group of histidine acid phosphatases (HAPs) that feature the conserved active site hepta-peptide motif RHGXRXP and the catalytically active dipeptide HD.[1] This group of phytases catalyzes phytic acid hydrolysis in a two-step mechanism via a nucleophilic attack from the histidine in the active site of 704 the enzyme to the scissile phosphoester bond of phytic acid In general, fungal phytases (E.C 3.1.3.8) initiate the splitting of the phosphate group at the C1 or C3 carbon of the inositol ring, and are thus called 3-phytases, whereas plant phytases (E.C 3.1.3.26) act preferentially at the C6 carbon, and are named 6-phytase However, phytases isolated from Escherichia coli, Lupinus albus, or Peniophora lycii are exceptions to this rule Interestingly, a soybean phytase is a purple acid phosphatase with a dinuclear iron iron or iron zinc center in the active site The phytase from Bacillus subtilis has a six-bladed folding scaffold, and calcium ion can affect its thermostability and catalysis As a whole, phytases show a strong ability to cleave equatorial phosphate groups, but a limited ability to hydrolyze axial phosphate groups The optimum pH for most known phytases is in the range of 4.5 Exceptions are phytases from mung bean, Enterobacter sp., or B subtilis that have their pH optimum in the neutral to alkaline range The temperature optimum of most plant and microbial phytases ranges from 45 65°C, higher than body temperatures of animals (37 40°C) Phytase activity unit is defined by the amount of inorganic phosphate released per minute from a selected substrate under certain pH and temperature In the case of A niger PhyA, the activity is determined in 0.05 0.2 M citrate or acetate buffer, pH 5.5, at 37°C; and one unit equals the amount of enzyme that releases mmol of inorganic phosphorus per minute from sodium phytate The biochemical properties of the currently available phytases for animal feeding are summarized in Table SUBSTRATE OCCURRENCE Phytases from A niger, Aspergillus terreus, E coli or Bacillus sp seem to have a high specificity for phytic acid, whereas plant phytases and some fungal enzymes such as the one from Aspergillus fumigatus have a broader substrate specificity Chemically, phytic acid refers to myoinositol-1,2,3,4,5,6-hexakis dihydrogen phosphate, which contains approximately 30% phosphorus Phytate and phytin refer to salts of phytic acid with individual or mixed Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019747 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Phytases 705 Fig Hydrolysis of phytate by phytase into inositol and phosphate Phytate hydrolysis also releases chelated metals such as iron, zinc, and calcium metals such as calcium, sodium, potassium, magnesium, iron, zinc, copper, etc In reality, all three of these compounds are indistinguishably called phytate Total contents of phytate are 0.5 1.9% in cereals, 0.4 2.1% in legumes, 2.0 5.2% in oil seeds, and 0.4 7.5% in protein products.[4] Distribution of phytate varies with seeds It is located in the germ of corn, in crystalloid-type globoids inside the protein bodies within the cotyledons of dicotyledoneous seeds (beans, soy, nuts, peanuts), or in the globoids of aleurone grains (protein bodies) present in the aleurone or bran layer of monocotyledoneous seeds (wheat, rice).[4] Phytate rapidly accumulates in seed ripening and serves mainly as storage of phosphorus, inositol, and minerals for the germinating seed It may be involved in the control of inorganic phosphate levels in both developing seeds and seedlings, and it may also have antifungic and antioxidant roles.[1,4] Because of these functions of phytate and its universal abundance in all plants, caution should be given in developing low-phytate crops However, phytate is an antinutrient factor in animal diets With a poor availability of phosphorus, phytate also chelates divalent metals such as zinc and iron There are twelve replaceable protons present in the phytic acid molecule: Six are dissociated in the strong acid range, one in the weak acid range, two with pK 6.8 to 7.6, and three with pK >10.[5] At the neutral pH of small intestine, phytic acid is strongly negatively charged and is able to complex or bind to positively charged molecules These complexes are rather insoluble, rendering the chelated metals unavailable for absorption NUTRITIONAL AND ENVIRONMENTAL BENEFITS Numerous studies have demonstrated the effectiveness of microbial or plant phytase added to plant-based diets for swine, poultry, and fish in improving utilization of phytate-phosphorus and reducing phosphorus excretion by these animals.[6] The efficacy of different phytases varies, but the average amount of phytase needed to replace g of inorganic phosphorus per kg of swine or poultry diet ranges from 500 to 1000 units.[7] With this efficacy, phytase can obviate inorganic phosphorus supplementation at least by half, saving the nonrenewable resource that may be exhausted in 80 years at the current extraction rate More urgently, supplemental phytase reduces fecal phosphorus excretion by 30 50%, which can potentially eliminate 90,000 tons of phosphorus excreted to the environment by poultry and swine in the United States annually In addition, phytase improves bioavailability of calcium, zinc, and iron, primarily by releasing these elements from binding to the phosphate groups of phytate However, the effects of phytase on utilization of protein, amino acids, or energy are still controversial DIETARY DETERMINANTS OF EFFICACY At least four dietary factors can modulate phytase efficacy First, high levels of dietary calcium or calcium/ phosphorus ratios reduce the effectiveness of phytase In phytase-supplemented diets, the recommended calcium/ phosphorus ratio is 1.2:1, not 2:1 as used in diets with adequate inorganic phosphorus added Second, moderate to high levels of inorganic phosphorus may inhibit the full function of phytase Third, supplemental organic acids such as citric acid or lactic acid enhance phytase efficacy Those acids may reduce the pH of stomach digesta, thus providing a better environment for phytase to function, and/or to enhance the solubility of digesta phosphorus and modify the transit time of digesta in the small intestine 706 Phytases Table Biochemical properties of phytases currently available for use in animal diets pH optimum Temperature optimum (°C) 2.5 3; 5.5 2.5 6.5 4.5 2.5 4.5 55 60 63 58 70 50 55 55 60 Bacillus sp.q,r,s,t,u 9.5 55 65 Wheatv,w 5.2 50 Origin A niger PhyAc,d,e,f A A P E niger PhyBg fumigatus PhyAe,h,i,j,k lyciil,m coli AppAh,i,n,o,p,w PI Mr (kDa)a Km (m M)b m Kcat (sÀ1)b Kcat/Km (sÀ MÀ 1)b 4.94 66 120 (50) 27 348 1.3 Â 107 7.04 7.3 3.61 4.37 6.3; 6.5 269 (65) 60 76 (49) 71 72 (44.6) 42 55 (45 48) 38 47 103 30 33 130 (IP6) 15 (IP5) 50 628 46 2200 6209 (IP6) 6926 (IP5) 26.6 6.1 Â 106 1.5 Â 106 6.6 Â 107 4.8 Â 107 (IP6) Â 108 (IP5) 5.3 Â 105 47 65 228 300 468 1.8 Â 106 5.0 5.1; 6.5 6.8 a The values in parentheses are calculated after deglycosylation of proteins or based on the deduced peptide sequence The value shown for A niger phyB is the molecular mass of the tetramer The molecular mass of the monomer is shown within parentheses b Only phytic acid (IP6) is used as the substrate for all enzymes except for E coli AppA Assay conditions are as follows: A niger PhyA: 58°C, pH 5.0; A niger PhyB: 63°C, pH 2.5; A fumigatus PhyA: 58°C, pH 5.0; P lycii: 58°C, pH 5.0; E coli AppA: 35 37°C, pH 4.5; Bacillus sp: 37°C, pH 7.0; Wheat: 55°C, pH 5.15; 35°C, pH 5.0 c Ullah et al (1999) Biochem Bioph Res Co 264: 210 206 d Han et al (1999) Appl Environ Microbiol 65: 1915 1918 e Ullah et al (2000) Biochem Bioph Res Co 275: 279 285 f Ullah et al (2002) Biochem Bioph Res Co 290: 1343 1348 g Ullah AHJ and Sethumadhavan K (1998) Biochem Bioph Res Co 243: 458 462 h Wyss et al (1999) Appl Environ Microbiol 65: 359 366 i Wyss et al (1999) Appl Environ Microbiol 65: 367 373 j Mullaney et al (2000) Biochem Bioph Res Co 275: 759 763 k Rodriguez et al (2000) Biochem Bioph Res Co 268: 373 378 l Lassen et al (2001) Appl Environ Microbiol 67: 4701 4707 m Ullah AHJ and Sethumadhavan K (2003) Biochem Bioph Res Co 303: 46 468 n Greiner et al (1993) Arch Biochem Biophys 303: 107 113 o Golovan et al (2000) Can J Microbiol 46: 59 71 p Rodriguez et al (2000) Arch Biochem Biophys 382: 105 112 q Kerovuo et al (1998) Appl Environ Microbiol 64: 2079 2085 r Kim et al (1998) FEMS Microbiol Lett 162: 185 191 s Kim et al (1998) Enz Microb Tech 22: t Choi et al (2001) J Prot Chem 20: 287 292 u Tye et al (2002) Appl Microbiol Biotechnol 59: 190 197 v Peers (1953) Biochem J 53: 102 110 w Greiner et al (2000) J Biotech 84: 53 62 In addition, organic acids may release cations chelated by phytate, reducing the amount of insoluble phytate cation complexes that are resistant to phytase action, thereby increasing the efficacy of endogenous or supplemented phytase Last, inclusion of hydroxylated cholecalciferol compounds has been shown to improve dietary phosphorus and zinc utilization by chicks in an additive manner with phytase Supplementing different phytases in combination has not shown any benefit over the singular additions However, adding phytase with other hydrolytic enzymes seems to produce a synergism Furthermore, there are several physical forms of phytase: powder, granule, and liquid The chemical coating of phytase to improve heat stability may somewhat compromise its release in stomach STORAGE AND HANDLING Phytase should be stored under dark, cool, and dry conditions When this is done, the enzyme may maintain good stability for months Refrigeration or freezing may extend its shelf life, whereas high storage temperature certainly decreases its activity Caution should be given in storing phytase mixed with vitamin and mineral premixes, as some of their components may have deleterious effects on phytase stability There are few reports on immune responses of workers who have inhalation exposure to phytase The hypersensitivity symptoms can be alleviated or avoided by implementing local exhaust systems and wearing protective clothing and masks with P2 filters.[8] Phytases DEVELOPING IDEAL PHYTASES A phytase would be considered ideal for feed application if it were catalytically effective, proteolysis-resistant, thermostable, and cheap The catalytic efficiency and protease susceptibility of any given phytase decide its ability to release phytate-phosphorus in the digestive tract The thermostability of phytase determines its feasibility in feed pelleting, and the overall cost to produce the enzyme ranks its final acceptance by industry Although there are significant differences in these features among various naturally occurring phytases, no single wild-type enzyme possesses all of the desired properties With advances of biotechnology, there are three ways to develop effective phytases with improved properties First, site-directed mutagenesis, based on crystal structure of phytases, has been applied to improve pH profile, thermostability, and catalytic efficiency Second, synthetic phytases such as the experimental consensus phytase have been generated based on homologous sequences of multiple phytases Last, new phytases can be produced by directed evolution with efficient selections A number of heterologous expression systems have been used for phytase production The expression hosts include plants, bacteria, fungi, and yeast.[2,9] Recently, transgenic pigs overexpressing a bacterial phytase in salivary gland have been generated.[10] If approved by regulatory agencies, this approach may serve as a sustainable and economical delivery of phytase 707 improvements in its property and reductions in its cost are warranted Modern biotechnology has provided great potential to develop ideal phytases and effective deliveries for specific groups of animals REFERENCES CONCLUSION There is an increasing need for phytase to improve dietary phytate-phosphorus utilization by livestock, and thus reduce their phosphorus excretion to the environment worldwide, in particular in areas of intensive animal production Although microbial phytase supplementation has been a widespread practice in swine and poultry feeding, and to a lesser extent, in fish feeding, continuous 10 Mullaney, E.J.; Daly, C.B.; Ullah, A.B.J Advances in phytase research Adv Appl Microbiol 2000, 47, 157 199 Lei, X.G.; Stahl, C.H Biotechnological development of effective phytases for mineral nutrition and environmental protection Appl Microbiol Biotechnol 2001, 57, 474 481 Liu, B.; Rafiq, A.; Tzeng, Y.; Rob, A The induction and characterization of phytase and beyond Enzyme Microb Technol 1998, 22, 415 424 Reddy, N.R.; Sathe, S.K.; Salukhe, D.K Phytates in legumes and cereals Adv Food Res 1982, 28, 92 (Academic Press, New York, NY) Cheryan, M Phytic acid interactions in food systems CRC Crit Rev Food Sci Nutr 1980, 13, 297 336 Lei, X.G.; Stahl, C.H Nutritional benefits of phytase and dietary determinants of its efficacy J Appl Anim Res 2000, 17, 97 112 Kornegay, E.T Chapter 18: Nutritional, Environmental, and Economic Considerations for Using Phytase in Pig and Poultry Diets In Nutrient Management of Food Animals to Enhance and Protect the Environment; Kornegay, E.T., Ed.; CRC, Lewis Publishing: New York, NY, 1996; 277 302 Baur, X.; Melching Kollmuss, S.; Koops, F.; Straburger, K.; Zober, A IgE mediated allergy to phytase A new animal feed additive Allergy 2002, 57, 943 945 Pandey, A.; Szakacs, G.; Soccol, C.R.; Rodriguez Leon, J.A.; Soccol, V.T Production, purification and properties of microbial phytases Bioresour Technol 2001, 77, 203 214 Golovan, S.P.; Meidinger, R.; 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.; Hackler, R.R.; Phillips, J.P.; Forsberg, C.W Pigs expressing salivary phytase produce low phosphorus manure Nat Biotechnol 2001, 19, 741 745 Poultry Production: Manure and Wastewater Management 745 Table Physicochemical properties of poultry manure Layerb Componenta Moisture (%) Weight (kg) Total solids (kg) Volatile solids (kg) Nitrogen (kg) Phosphorus (kg) Potassium (kg) Broiler Turkey As excreted As removed As excreted As removed As excreted As removed 75 27.5 6.9 4.9 0.38 0.14 0.15 50 10.9 5.5 75 36.4 9.1 6.8 0.5 0.15 0.21 24 15.9 12.0 9.7 0.31 0.15 0.18 75 19.8 5.0 4.4 0.34 0.13 0.13 34 11.0 7.3 0.19 0.13 0.14 0.40 0.18 0.20 No data Estimates based on 455 kg (1000 lb) live weight per day for all bird types b High rise layer housing only No bedding added to layer manure (Adopted from Ref 2.) a litter In some parts of the United States, a built-up litterbased system may be used for two to three years before a complete clean out The litter is land-applied for fertilizing crops and pastures immediately after removal or is stored for later application As shown in Fig 1, mortality from these poultry production systems may be handled using on-site incineration or composting (usually with litter as a co-composting material) The finished compost is then land-applied as fertilizer Off-site disposal is accomplished through rendering Burial of dead birds is prohibited in many states, but may be allowed if a large number of mortalities occur as a result of catastrophes POULTRY MANURE TREATMENT AND UTILIZATION Most poultry manure is used for crop and pasture fertilization as a cost-effective alternative to inorganic mineral fertilizer Land application also recycles nutrients, enhances soil fertility, and improves soil physical properties However, a balance must be maintained between maximum utilization of nutrients by crops and the risk of health and environmental impacts Proper managing of poultry manure from its production through utilization is the key to maintaining this balance This includes proper design and siting of housing, manure storage, and mortality management facilities; and comprehensive nutrient management planning Education and training of managers and operators of poultry production systems are essential for good manure management Manure and soils receiving manure should be tested for available nutrients before application Application rates have typically been based on N requirements of crop For soils testing high in P, manure application should be based on the crop’s P requirements Manure application that exceeds a crop’s ability to take up N may threaten water quality Nitrogen as nitrate is a highly mobile compound that may cause human and animal health problems if drinking water concentrations are greater than 10 mg/l Soil P enrichment occurs as a result of overfertilization with P Phosphorus applied to fields as inorganic fertilizer or from manure can move into bodies of water through erosion and runoff events and can accelerate eutrophication (the natural aging process of lakes and streams), leading to excessive algae growth, oxygen deficiency, and fish mortality Some of the liquid or solid manure may be put to alternative uses, with or without undergoing a treatment process Liquid manure from layer operations is sometimes stored and treated in anaerobic (oxygen-free) lagoons and further diluted with additional water, while anaerobic bacteria biodegrade volatile organic compounds Part of the total solids in manure are settled as sludge at the lagoon bottom, while the supernatant (the liquid standing above suspended solids and sludge) can be recycled for flushing layer houses and irrigated as a source of nutrient and water for plants Liquid poultry manure may also be anaerobically digested in insulated, airtight containers (digesters) to produce biogas (methane and carbon dioxide) The methane gas produced with this treatment process is combustible and can be harvested to produce energy This energy can be converted to electricity The closed biogas digesters also control manure odors A small portion of the litter and straw, hay, or crop residue may be used as a carbon source for animal mortality composting, with the resulting compost used as fertilizer In some parts of the United States, broiler litter or dried layer manure is used in the microbial mixture to supply nutrients for Agaricus mushrooms.[4] 746 Poultry Production: Manure and Wastewater Management Fig Waste storage management and utilization options for poultry operations (Adapted from USDA NRCS Agricultural Waste Management Field Handbook, Part 651.) (View this art in color at www.dekker.com.) Direct incineration of raw broiler litter with high ash content (soil and other incombustibles in the litter) in conventional furnaces has proven difficult due to incomplete combustion, slag formation, odor, gas, and particulate emissions to the environment On a commercial scale, electric power generation plants in the United Kingdom are firing poultry litter as a furnace fuel to generate boiler steam The ash produced from litter combustion is recovered and sold as nutrient-rich fertilizer.[5] The feasibility of cofiring biomass (poultry litter and beef feedlot manure) with coal as a fuel for energy Poultry Production: Manure and Wastewater Management generation is currently being studied in the United States.[6,7] Preliminary results from small-scale boiler experiments show that blends of coal and biomass can be successfully fired, and that nitrogen oxide pollutant emissions were similar to or lower than those from firing coal only CONCLUSION In the United Sates, poultry production operations have increased in size and are regionally concentrated Poultry manure is managed as liquid or solid, depending on the manure management system and bird type Therefore, physicochemical properties of poultry manure constituents (N, P, and K, etc.) vary from within species, and among bird types When properly managed, poultry manure provides beneficial nutrients for plant growth, and improves soil quality without harming the natural environment The majority of poultry manure and wastewater is land-applied as organic fertilizer A small portion of poultry manure is treated or processed for alternative uses, such as biogas and compost for specialty crops Incineration of litter to produce electricity is being carried out in the United Kingdom Co-combustion of poultry litter with coal is currently being investigated 747 REFERENCES Lorimor, J.; Powers, W.; Sutton, A Manure Characteristics, MWPS 18, Section1; Midwest Plan Service: Ames, IA, 2000; 23 Agricultural Waste Characteristics, Agricultural Waste Management Field Handbook, Part 651; U.S Department of Agriculture Natural Resources Conservation Service: Washington, DC, 1992 Agricultural Charts and Maps; U.S Department of Agriculture National Agricultural Statistics Service: Washington, DC, 2000 http://www.usda.gov/nass/ aggraphs/graphics.htm (accessed October, 2003) Collins, E.R., Jr.; Barker, J.C.; Carr, L.E.; Brodie, H.L.; Martin, J.H., Jr Poultry Waste Management Handbook; Natural Resource, Agricultural, and Engineering Service: Ithaca, NY, 1999; 64.6 NRAES 132 Fibrowatt Limited: London, United Kingdom http:// www.fibrowatt.com/UK Corporate/index.html (accessed October 2003) Sami, M.; Annamalai, K.; Woldridge, M Co firing of coal and biomass fuel blends Prog Energy Combust Sci J 2001, 27, 171 214 Annamalai, K.; Sweeten, J.; Mukhtar, S.; Thien, B.; Wei, G.; Piryadarsan, S.; Arumugam, S.; Heflin, K Co Firing Coal: 2003 Feedlot and Litter Biomass (CFB and CLB) Fuels in Pulverized Fuel and Fixed Bed Burners Final Report Submitted to the U.S Department of Energy Grant number: DE FG26 00NT40810 Pregnancy: Maternal Response Alan W Bell Cornell University, Ithaca, New York, U.S.A INTRODUCTION Pregnancy imposes a metabolic burden on female mammals that increases with advancing gestation to a degree that is related directly to rate of conceptus growth scaled for maternal size To accommodate the increasing nutrient demands of the fetus(es) and nonfetal conceptus tissues, the dam makes a series of coordinated metabolic adjustments in various nonuterine tissues to ensure that fetal nutrition is relatively unaffected by moderate variations in maternal nutrient intake The homeorhetic regulation and coordination of these adaptations will be discussed Most examples are from domestic ruminants but the general principles apply to other mammalian species Mechanisms appear to involve reduced expression and, possibly, altered intracellular translocation, of the insulinresponsive glucose-transport protein, GLUT4, in muscle and adipose tissues.[4] The increase in glucose availability created by the combination of increased hepatic production and reduced utilization in peripheral tissues is of advantage to the pregnant uterus, as well as to vital maternal tissues such as brain, because these tissues not require insulin to facilitate the uptake and transport of glucose Thus, a relatively normal supply of maternal glucose to the fetus can be sustained even if the dam is somewhat undernourished, especially if she has recourse to adequate lipid stores in adipose tissue.[5] Lipid Metabolism METABOLIC ADAPTATIONS IN MATERNAL TISSUES Glucose Metabolism Some of the increase in hepatic gluconeogenesis in latepregnant ruminants is due to increased voluntary feed intake.[1] However, intake is often constrained by physical factors such as diet quality and abdominal distension, as well as endocrine factors such as the surge in estrogen secretion in late pregnancy.[2] Under these or more controlled conditions of feed restriction, an increase in glucose production is sustained by increased peripheral mobilization and hepatic uptake of endogenous substrates such as amino acids from skeletal muscle and glycerol from the lipolysis of adipose triglycerides.[1,3] Glucose utilization by muscle and adipose tissue tends to decrease during late pregnancy, especially if maternal energy intake is restricted or voluntarily declines These tissues account for most of the approximately 20% of total glucose disposal that, in nonpregnant, nonlactating sheep, appears to be insulin-dependent It is therefore likely that altered responses to insulin in these tissues largely account for the development of moderate insulin resistance in various parameters of whole-body glucose disposal in late-pregnant ewes.[1] This effect, which presumably mediates the so-called glucose-sparing effect of pregnancy, is exaggerated by moderate maternal undernutrition 748 There is limited evidence that the lipogenic capacity of adipose tissue is enhanced during early midpregnancy in ruminants, possibly mediated by increased tissue response to insulin.[6] However, these observations are confounded in cattle by the overlapping of pregnancy and lactation, and in sheep by the possible influence of season on voluntary feed intake.[7] Even if there is not a specific enhancement of lipid synthesis and deposition during early pregnancy, there is no doubt that at this stage the well-fed animal can efficiently take advantage of adequate energy intake to deposit lipid stores in anticipation of the increased energy demands of late pregnancy and, especially, of early lactation During late pregnancy, adipose tissue becomes refractory to the lipogenic and antilipolytic influences of insulin and more sensitive to the lipolytic effects of adrenergic agents.[6] At least part of this increased propensity for fat mobilization is independent of energy intake and appears to be part of the regulated physiological prelude to lactation, but the general response is markedly exaggerated by maternal undernutrition.[7] In ruminants and pigs, at least, placental impermeability to nonesterified fatty acids (NEFA) prevents direct fetal access to this source of maternal stored energy However, increased NEFA uptake and oxidation in maternal tissues allows the sparing of glucose for use in conceptus tissues as described previously Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019768 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Pregnancy: Maternal Response 749 Nitrogen Metabolism Effects of pregnancy on maternal nitrogen metabolism have been less studied than those on carbohydrate and lipid supply and utilization Increased predisposition to the mobilization of amino acids from skeletal muscle during late pregnancy can be inferred from measurements of tissue nitrogen balance in ewes.[8] This response may be permitted by the waning influences of insulin and other anabolic factors, including insulin-like growth factor (IGF-1) as term approaches.[9] Coincidentally, hepatic protein synthesis is upregulated in dairy cows at a time when voluntary intake of dry matter and dietary protein is declining in the prepartum period.[10] The dam also must dispose of the significant increase in products of nitrogen catabolism generated by conceptus tissues during late pregnancy Fetal urea synthesis is substantial because of extensive oxidative deamination of amino acids in fetal liver and other tissues This urea is efficiently transported by the placenta to the maternal circulation and adds directly to maternal urea destined for renal clearance and excretion The placenta, which has little urea cycle enzyme activity, also deaminates amino acids to an extent that causes perceptible increases in maternal blood-ammonia concentrations and the need for hepatic detoxification.[3] REGULATION AND COORDINATION OF METABOLIC ADAPTATIONS Homeorhetic Regulation of Metabolic Adaptations to Pregnancy The concept of homeorhesis as it applies to regulation of nutrient partitioning was elaborated by Bauman and Currie[11] and, more recently, Bauman.[12] Examples of homeorhetic regulation in pregnant animals have been reviewed previously.[1,4] Several pregnancy-related hormones, including progesterone, estradiol, and placental lactogen (PL) have been suggested as homeorhetic modulators of observed changes in tissue responses to insulin and catecholamines, and associated metabolic adaptations to the state of pregnancy in ruminants.[1] A more recently suggested candidate is leptin,[4] adipose tissue expression and plasma concentration of which increase markedly in ewes during midpregnancy, independent of nutrition and energy balance These hormones and their proposed actions are listed in Table None of these putative regulators has been shown to have the integrative, pleiotropic influences that growth hormone (GH) has in lactating ruminants.[12,13] Possibly, the combined influence of these hormones is more significant than their varying individual influences at different stages of pregnancy Among the sex steroids, estradiol-17b may contribute directly or indirectly to mediation of some metabolic adaptations, especially close to term when there is a pronounced surge in plasma estrogen concentrations Treatment of ovariectomized ewes with estradiol caused a reduction in rates of adipose lipogenesis and fatty acid re-esterification However, we were unable to discern any effect of a similar hormonal treatment on responses of glucose or NEFA metabolism in vivo to insulin or catecholamines, although basal plasma concentrations of glucose, NEFA, and glycerol were chronically elevated in treated animals.[3] Estradiol also may contribute indirectly to changes in lipid metabolism through its inhibitory effect on voluntary feed intake in late-pregnant ruminants.[2] Definitive evidence of a homeorhetic role for PL remains elusive, but such a putative role is hard to dismiss, Table Possible homeorhetic hormones and their proposed actions on tissues during pregnancy State Mid pregnancy Hormone Progesterone Leptin Putative action " Insulin sensitivity # Catecholamine sensitivity Late pregnancy Placental lactogen Estrogens # Insulin sensitivity and responsiveness " Catecholamine sensitivity and responsiveness Tissue/response " Adipose glucose uptake " Adipose lipogenesis # Adipose lipolysis " Liver gluconeogenesis # Glucose uptake by adipose and muscle # Adipose lipogenesis # Muscle amino acid uptake and protein synthesis " Muscle proteolysis " Adipose lipolysis 750 as we have discussed elsewhere.[3] Indirect evidence for such a role includes its cross-reactivity with both GH and prolactin receptors in maternal ruminant tissues and its increased specific binding in a likely target, adipose tissue, as pregnancy advances Cross-reactivity with the GH receptor would be consistent with the development of insulin resistance in adipose tissue since GH is a potent homeorhetic effector of this response in ruminant adipose tissue.[14] Also, moderate undernutrition enhances placental gene expression and secretion of PL in late-pregnant ewes, coincident with the decreased expression of GLUT-4 in maternal insulin-responsive tissues and exaggeration of indices of whole-body insulin resistance.[4] The apparently pregnancy-specific increase in leptin expression and secretion by adipose tissue in sheep, together with increasing evidence that leptin modulates the metabolic actions of insulin in rodents, suggests that this peptide should be added to the list of putative homeorhetic effectors of metabolic adaptations to pregnancy In addition, the abundant placental expression of the physiologically relevant OB-Rb form of the leptin receptor suggests that leptin may act as a direct signal of maternal energy balance to the placenta.[3] Finally, it must be recognized that maternal adaptations to pregnancy not occur independently from metabolic and endocrine development of the conceptus The interplay and coordination of maternal and fetal influences is the focus of another contribution to this volume Pregnancy: Maternal Response REFERENCES CONCLUSIONS Pregnancy-induced adaptations in the metabolism of glucose, lipids, and, to a lesser extent, amino acids have been well described in ruminants and other domestic animals These adaptations are mediated by altered responses in multiple tissues to homeostatic effectors such as insulin and catecholamines in a way that is coordinated to promote the availability of vital nutrients to the developing fetus(es) These phenomena are the hallmarks of homeorhetic regulation of nutrient partitioning in support of a chronic physiological imperative However, the identity of the factor(s) responsible for homeorhetic modulation of metabolic adaptations to pregnancy remains to be elucidated 10 11 12 13 ARTICLE OF FURTHER INTEREST 14 Female Reproduction: Maternal Fetal Relationship, p 408 Bell, A.W.; Bauman, D.E Adaptations of glucose metabolism during pregnancy and lactation J Mamm Gland Biol Neopl 1997, (3), 265 278 Forbes, J.M The Effects of Sex Hormones, Pregnancy, and Lactation on Digestion, Metabolism and Voluntary Intake In Control of Digestion and Metabolism in Ruminants; Milligan, L.P., Grovum, W.L., Dobson, A., Eds.; Prentice Hall: Englewood Cliffs, USA, 1986; 420 435 Bell, A.W.; Ferrell, C.L.; Freetly, H.C Pregnancy and Fetal Metabolism In Quantitative Aspects of Ruminant Digestion and Metabolism, 2nd Ed.; Dijkstra, J., Forbes, J.M., France, J., Eds.; CABI Publishing: Wallingford, U.K., 2004 (in press) Bell, A.W.; Ehrhardt, R.A Regulation of Macronutrient Partitioning Between Maternal and Conceptus Tissues in the Pregnant Ruminant In Ruminant Physiology Diges tion, Metabolism, Growth and Reproduction; Cronje, P.B., ´ Ed.; CABI Publishing: Wallingford, U.K., 2000; 275 293 Bell, A.W Pregnancy and Fetal Metabolism In Quantita tive Aspects of Ruminant Digestion and Metabolism; Forbes, J.M., France, J., Eds.; CAB International: Wall ingford, U.K., 1993; 405 431 Vernon, R.G.; Sasaki, S Control of Responsiveness of Tissues to Hormones In Physiological Aspects of Diges tion and Metabolism in Ruminants; Tsuda, T., Sasaki, Y., Kawashima, R., Eds.; Academic Press: San Diego, 1991; 155 182 Bell, A.W.; Bauman, D.E Animal Models for the Study of Adipose Regulation in Pregnancy and Lactation In Nutrient Regulation During Pregnancy, Lactation, and Infant Growth; Allen, L., King, J., Lonnerdal, B., Eds.; ă Plenum Press: New York, 1994; 71 84 McNeill, D.M.; Slepetis, R.; Ehrhardt, R.A.; Smith, D.M.; Bell, A.W Protein requirements of sheep in late pregnan cy: Partitioning of nitrogen between gravid uterus and maternal tissues J Anim Sci 1997, 75 (3), 809 816 Bell, A.W.; Burhans, W.S.; Overton, T.R Protein nutrition in late pregnancy, maternal protein reserves and lactation performance in dairy cows Proc Nutr Soc 2000, 59 (1), 119 126 Bell, A.W Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation J Anim Sci 1995, 73 (9), 2804 2819 Bauman, D.E.; Currie, W.B Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving homeostasis and homeorhesis J Dairy Sci 1980, 63 (9), 1514 1529 Bauman, D.E Regulation of Nutrient Partitioning During Lactation: Homeostasis and Homeorhesis Revisited In Ruminant Physiology Digestion, Metabolism, Growth and Reproduction; Cronje, P.B., Ed.; CABI Publishing: Wall ´ ingford, U.K., 2000; 311 328 Bauman, D.E.; Vernon, R.G Effects of exogenous bovine somatotropin on lactation Annu Rev Nutr 1993, 13, 437 461 Etherton, T.D.; Bauman, D.E Biology of somatotropin in growth and lactation of domestic animals Physiol Rev 1998, 78, 745 761 Pregnancy: Recognition/Signaling M P Green R M Roberts University of Missouri, Columbia, Missouri, U.S.A INTRODUCTION If a pregnancy is to be established successfully, a conceptus must signal its presence to the maternal system, which in turn must adjust her physiology to the needs of her developing offspring This series of events is often termed maternal recognition of pregnancy (MRP) One crucial aspect of MRP in the domestic livestock species is that ovarian cyclicity must be interrupted and corpus luteum (CL) lifespan extended, although the embryo most likely makes its presence known to the mother well before it intervenes in events of the estrous cycle by releasing bioactive compounds ‘‘sensed’’ by local maternal tissues However, embryo transfer can be completed successfully until quite late in development, suggesting that the very early signals are relatively unimportant to the long-term effects on pregnancy outcome CL MAINTENANCE The timing and strength of embryonic signals probably allow the mother to evaluate the fitness of a conceptus and to terminate a pregnancy before investment is too high Embryos that possess chromosomal abnormalities, that are developmentally stunted, or are suffering setbacks as the result of detrimental environmental and nutritional conditions in the uterus, are likely to be delayed in their development and to signal less than robustly to the mother.[1,2] Accordingly, deficient MRP signaling probably underpins much of the early embryonic loss that occurs in the first three weeks of pregnancy In early pregnancy, the CL is the major source of progesterone, a steroid hormone that acts on the endometrium to maintain it in a state whereby it remains receptive and provides nourishment to the embryo.[3,4] Two strategies are principally employed by conceptuses to avoid a return to cyclicity: the production of either a luteotrophic signal that promotes CL growth and activity or an antiluteolytic signal that protects the CL.[3] There are examples, e.g., dogs and marsupials, where no intervention is needed because the duration of pregnancy corresponds to the length of the luteal phase of the estrous Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019766 Copyright D 2005 by Marcel Dekker, Inc All rights reserved cycle The estrous cycles of livestock are short compared to pregnancy so that CL rescue is required.[2,5,6] In all livestock, luteolysis is initiated by prostaglandin F2a (PGF2a) produced in the endometrium (the lining of the uterus) toward the end of the cycle.[7] PGF2a reaches the ovary via maternal blood, and, by mechanisms still not completely clear, causes the structural and functional demise of the progesterone-producing luteal cells In some species, oxytocin has a role in initiating PGF2a release from the endometrium.[7] Conceptuses ensure CL lifespan extension by either intervening in the production and/or release of PGF2a, counteracting its actions at the level of the ovary, or by a combination of strategies.[3,9] BOVIDAE (CATTLE, SHEEP, GOAT, AND THEIR RELATIVES) In these species, the trophectoderm (outer) cells of the conceptus begin to produce an antiluteolytic protein factor called interferon-tau (IFN-t) at the blastocyst stage of development Synthesis becomes maximal as the conceptus elongates in the period immediately preceding attachment of trophectoderm to the uterine wall.[5] The progenitor IFN-t gene duplicated from an IFN-o gene approximately 36 million years ago (MYA) As a consequence, this gene family is unique to the Ruminantia suborder.[9] IFN-t is expressed prior to the release of luteolytic pulses of PGF2a from the endometrium[10] and abrogates PGF2a release by downregulating transcription of the estrogen and oxytocin receptor genes in the uterine endometrium.[4,6,8,10] IFN-t may simultaneously promote production of prostaglandin E2 (PGE2), which has a luteotrophic rather than luteolytic action on the CL and exerts a local immunosuppressive action at the maternal conceptus interface.[5] CAMELIDS The camelids (camels, llamas and alpacas) diverged from the lineage leading to the true ruminant species at least 751 752 40 MYA.[9] The female camel is a seasonal breeder and only ovulates in response to mating The CL develops within a few days, functionally plateaus around day postcoitum, and then promptly regresses.[11] Thus the conceptus must initiate its signal within about a week of conception if the pregnancy is to be maintained In nonpregnant dromedary camels, basal concentrations of PGF2a rise and progesterone levels fall after day of the cycle, thereby implicating PGF2a as the luteolytic factor, although oxytocin appears not to be involved The nature of the antiluteolytic signal remains unknown, but it is almost certainly not IFN-t SUIDAE (PIGS AND THEIR RELATIVES) The pig lineage diverged earlier than the camelids, about 55 MYA.[9] PGF2a is central to the luteolytic process and can be used pharmacologically to regress pig CL to induce estrus.[3] Although pig conceptuses produce at least two forms of IFN (IFN-g and IFN-d), these appear not to be important in counteracting PGF2a but probably have a local effect on the maternal immune system.[2] Instead, the unattached elongating porcine conceptuses produce large amounts of estrogen beginning about day 10 of pregnancy.[2] Estrogen increases endometrial receptors for prolactin, which cause the uterine epithelial cells to redirect their release of PGF2a into the uterine lumen rather than basolaterally into the maternal vasculature toward the ovary and CL Consequently, estrogen injections on days 11 to 15 of the cycle can induce a prolonged pseudopregnancy.[5] PGE2 is also produced and may act as a luteoprotective/luteotrophic agent.[2] Doubts remain, however, as to whether the estrogen-based mechanism is the sole mechanism that prevents luteolysis in the pig.[12] EQUIDS (HORSES AND THEIR RELATIVES) The horse lineage diverged from the pig lineage about 80 MYA.[9] PGs synthesized by the early conceptus induce the muscular contractions that propel it from the oviduct into the uterus The equine conceptus is unusual in that it expands spherically rather than elongating and does not assume a stationary position in the uterus until about day 18.[13] Prior to this stage, it migrates through the uterine lumen 12 to 14 times a day Endometrial PGF2a is the trigger for luteolysis, but there is no firm evidence for either production of an IFN or a luteoprotective role of estrogen.[13] PGE2 production by the conceptus and uterus increases during this period and may play a role in CL maintenance If the migration of the conceptus through the Pregnancy: Recognition/Signaling uterine lumen is physically prevented, the CL is not protected Thus, intrauterine migration of the conceptus and possibly its direct contact with the uterine epithelium of both uterine horns is directly implicated in the suppression of luteolytic pulses of PGF2a APPLICATIONS AND IMPLICATIONS The compounds responsible for MRP have the potential ability to safeguard pregnancies that are at risk Intramuscular injections of IFN improved lambing rates and the numbers of lambs born in flocks where fertility was low, but were unhelpful in beef cattle.[14] On the other hand, IFN-t was successful in increasing pregnancy rates after asynchronous embryo transfers in red deer.[15] Consequently, boosting the MRP signal may be useful in rescuing developmentally normal, but otherwise delayed, embryos of intra- or interspecies pregnancies for the purpose of saving endangered species or in producing hybrid or cloned animals Finally, factors produced by the early placenta and released into the maternal bloodstream have the potential to be used as pregnancy tests.[16] CONCLUSIONS The production of PGF2a by the endometrium toward the end of the estrous cycle causes the CL to regress In pregnancy, CL lifespan is extended due to the release of conceptus factors that prevent luteolysis In short, establishment of pregnancy depends on the ability of the conceptus to elicit a signal and of the mother to respond appropriately This process remains poorly understood and differs across species In a few species, biologically relevant active factors released by the conceptus have been identified, while in others they remain unknown (Table 1) Because multiple signals undoubtedly pass between the conceptus and the mother, maternal responses are dynamic and complex Those MRP signals that ensure Table Summary of MRP signals and the days of recognition in domestic species Species Cattle Sheep Camels Pigs Horses a Assumed Days of recognition 14 12 11 12 17 15 8a 14 14 Main signal(s) IFN t IFN t Unknown Estrogen and prolactin Estrogena Pregnancy: Recognition/Signaling extension of CL lifespan, although crucial to continuance of the pregnancy, are only the beginning of a sequence of endocrine, paracrine, and autocrine signals that direct changes in maternal physiology and ensure a successful pregnancy REFERENCES Goff, A.K Embryonic signals and survival Reprod Domest Anim 2002, 37, 133 139 Bowen, J.A.; Burghardt, R.C Cellular mechanisms of implantation in domestic farm animals Cell Dev Biol 2000, 11, 93 104 Thatcher, W.W.; Bazer, F.W.; Sharp, D.C.; Roberts, R.M Interrelationship between uterus and conceptus to main tain corpus luteum function in early pregnancy: Sheep, cattle, pigs and horses J Anim Sci 1986, 62 (Suppl 2), 25 46 Spencer, T.E.; Bazer, F.W Biology of progesterone action during pregnancy recognition and maintenance of preg nancy Front Biosci 2002, 7, D1879 D1898 Roberts, R.M.; Xie, S C.; Mathialagan, N Maternal recognition of pregnancy Biol Reprod 1996, 54 (2), 294 302 Flint, A.P.F Interferon, the oxytocin receptor and the maternal recognition of pregnancy in ruminants and non ruminants: A comparative approach Reprod Fertil Dev 1995, (3), 313 318 McCracken, J.A.; Custer, E.E.; Lamsa, J.C Luteolysis: A neuroendocrine mediated event Physiol Rev 1999, 79 (2), 264 323 753 10 11 12 13 14 15 16 Mann, G.E.; Lamming, G.E.; Robinson, R.S.; Wathes, D.C The regulation of interferon t production and uterine hormone receptors during early pregnancy J Reprod Fertil Suppl 1999, 54, 317 328 Roberts, R.M.; Ealy, A.D.; Alexenko, A.P.; Han, C S.; Ezashi, T Trophoblast interferons Placenta 1999, 20, 259 264 Roberts, R.M.; Ezashi, T.; Rosenfeld, C.S.; Ealy, A.D.; Kubisch, H.M The interferon t: Evolution of the genes and their promoters, and maternal trophoblast interactions in control of their expression Reprod., Suppl 2003, 61, 239 251 Skidmore, L.; Adams, G.P Recent Advances in Camelid Reproduction; Intl Vet Info Service: Ithaca, NY, 2000 Zeicik, A.J Old, new and the newest concepts of inhibition of luteolysis during early pregnancy in the pig Domest Anim Endocrinol 2002, 23, 265 275 Allen, W.R.; Stewart, F Equine placentation Reprod Fertil Dev 2001, 13 (7 8), 623 634 Barros, C.M.; Newton, G.R.; Thatcher, W.W.; Drost, M.; Plante, C.; Hansen, P.J The effect of bovine interferon on pregnancy rate in heifers J Anim Sci 1992, 70 (5), 1471 1477 Demmers, K.J.; Jabbour, H.N.; Deakin, D.W.; Flint, A.P.F Production of interferon by red deer (Cervis elaphus) conceptuses and the effects of rIFN t on the timing of luteolysis and the success of asynchronous embryo transfer J Reprod Fertil 2000, 118 (2), 387 395 Humbolt, P Use of pregnancy specific proteins and progesterone assays to monitor pregnancy and deter mine the timing, frequencies and sources of embryonic mortality in ruminants Theriogenology 2001, 56, 1417 1433 Probiotics Stanley E Gilliland Oklahoma State University, Stillwater, Oklahoma, U.S.A INTRODUCTION The use of probiotics (also referred to as direct-fed microbials) in relation to feed supplements for animals was initiated in approximately 1974, although the suggestion for the use of such bacteria for the human diet dates much earlier In the early 1900s, Eli Metchnikoff advocated that humans should consume milk fermented with lactobacilli in order to displace the undesirable microorganisms that may occur in the intestinal tract Since the 1970s many feed supplements have been marketed as sources of probiotics Unfortunately, in the late 1970s many of these products contained few, if any, viable probiotic bacteria Thus, many reports in which probiotics were evaluated provided no conclusive evidence about their efficacy This was in part due to low viability of the microorganism in the products and was further complicated by the fact that the probiotic bacteria, especially the lactobacilli, exhibit host-specificity, which was not considered For example, one strain of Lactobacillus isolated from one animal species is not expected to function well in another animal species POTENTIAL BENEFITS Interest in the use of probiotics as livestock feed supplements is largely due to a concern over use of subtherapeutic levels of antibiotics in the livestock rations The earliest potential benefit advocated for using probiotic bacteria in the human diet was to exert control over intestinal flora It is assumed that the same type of relationship would occur in animals Thus the primary potential benefit of probiotics is to control intestinal infections in the livestock Some properly selected probiotic bacteria can also increase nutrient utilization by providing enzymes in the gut capable of converting certain components of the diet into more easily used nutrients for the host animal Some studies have suggested the possibility of feeding selected probiotic bacteria to produce certain changes in the body composition of the animals or their products such as altering the lipid composition The specific function of probiotics may be different depending on the host animal and, more important, on the characteristics of the probiotic Viability 754 of the probiotic at the time of consumption is considered very important However, the viability in feed supplements may be low.[1] BACTERIAL SPECIES INVOLVED Lactobacillus is the genus that contains most of the bacteria considered for use as probiotics for animals Included in this genus as potential probiotics are Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus reuteri Species of Bifidobacterium, as well as species of Propionibacterium, have also been tested and proposed as probiotics for certain animals Other species of microorganisms having potential include Streptococcus faecalis and species of Bacillus These groups of microorganisms represent the major ones that have been observed in the scientific literature related to probiotics for livestock It is very important to remember that one particular strain of one species of a given bacterial genus should not be expected to function in all species of animal or to provide all of the potential benefits for those animals.[3] Selection of the individual strains within species to be used is thus extremely important PROBIOTICS FOR POULTRY The most widely studied livestock species with respect to the use of probiotics is poultry Much of the attention in this area has been focused on the control of salmonella in chickens Properly selected cultures of probiotics (such as Lactobacillus species) can overcome those lactobacilli found in the natural flora of the birds and exert inhibitory action toward salmonella in the intestinal tract of chickens.[5] Another approach has been to culture the intestinal bacterial flora from a healthy chicken and to use this preparation to inoculate one-day-old chicks in order to establish a healthy normal flora, which helps control salmonella The problem with this approach is possible lack of consistency in the organisms making up the preparation from one batch to another Some refer to the use of probiotics in poultry to control pathogens as competitive exclusion However, the mechanism by Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019772 Copyright D 2005 by Marcel Dekker, Inc All rights reserved Probiotics 755 L acidophilus NPC 747, have also been shown to increase daily gain and feed efficiency in feedlot cattle.[4] Feeding a mixture of selected cultures of Propionibacterium and E faecium resulted in a trend toward reduced acidosis in feedlot cattle.[12] Feeding dairy cattle a probiotic containing E faecium plus a yeast culture resulted in increased milk yield.[13] Other studies also indicate that feeding selected cultures of L acidophilus resulted in increased milk yield in dairy cattle The exact mechanism of the benefits provided by the probiotics in improving feed efficiency, growth, and milk production has not been determined Fig Lactobacillus acidophilus NPC 747 is effective in reducing the frequency of occurrence of Escherichia coli O157:H7 in feedlot cattle PROBIOTICS FOR OTHER ANIMAL SPECIES which probiotic preparations are able to inhibit intestinal pathogens in poultry and in other animals has not been clearly defined With regard to nutrition and growth, feeding a selected culture of L acidophilus to laying hens increased egg production and feed conversion, and reduced the cholesterol level in the egg yolks.[6] Not much appears in the scientific literature concerning the evaluation of probiotics for other animal species Whereas there may be probiotic products available today for horses, fish, dogs, and cats, very little scientific research has been published on their efficacy For those products that are available, host specificity may not have been considered and carefully controlled experiments to evaluate them are lacking PROBIOTICS FOR SWINE CONCLUSIONS There is interest in the swine industry to find ways to control salmonella and/or other pathogens during the feeding phase.[7] Not much scientific research has been reported on this with regard to the potential for using probiotic cultures However, feeding a mixture of B pseudolongum and L acidophilus to piglets decreased the frequency of mortality.[8] In the same report these probiotic organisms also increased the weight gain of the piglets Lactobacilli are very prevalent in the duodenum, jejunum, and ileum of healthy piglets; they thus represent a part of the natural flora.[9] Feeding a culture of L acidophilus selected for amylase activity to weaning-age piglets on a high-starch diet resulted in increased growth and feed efficiency.[10] Currently, efforts are underway to establish whether or not a selective culture of L acidophilus would exert inhibitory action on salmonella in pigs Probiotics have the potential to provide a number of benefits for livestock as well as companion animals One strain of one species of a bacterial culture should not, however, be expected to provide all benefits The major group of bacteria considered for use as probiotics are in the genus Lactobacillus There are many naturally occurring variations in relative functional ability among strains of each individual species within this genus The same is true for other genera of the lactic acid bacteria Thus, strains to be used for probiotics should be carefully selected for the ability to provide the desired benefit in the host animal Host specificity is also very important for probiotics whose benefits require that they be able to grow and function in the intestinal tract Probiotics should be tolerant to bile and to other material, such as stomach acids, in the digestive system To be successfully marketed they should be easy to grow in commercial culture production facilities Additionally, probiotics must be able to survive production, processing, storage, and delivery to the animal PROBIOTICS FOR CATTLE A major benefit of feeding L acidophilus NPC 747 (Fig 1) to cattle has been a significant reduction in the frequency of occurrence of Escherichia coli O157:H7 in feedlot cattle.[11] This is considered a very important intervention step in the feedlot cattle industry to reduce the occurrence of this pathogen on fresh meat Probiotics, including REFERENCES Fuller, R History and Development of Probiotics In Probiotics the Scientific Basis, 1st Ed.; Chapman & Hall: New York, 1992; 756 Probiotics tration of bifidobacteria and lactic acid bacteria to newborn calves and piglets J Dairy Sci 1995, 78 (12), 2838 2846 Rojas, M.; Conway, P.L Colonization by lactobacilli of piglet intestinal mucus J Appl Bacteriol 1996, 81 (5), 474 480 Lee, H.S.; Gilliland, S.E.; Carter, S Amylolytic cultures of Lactobacillus acidophilus: Potential probiotics to improve dietary starch utilization J Food Sci 2001, 66 (2), 338 344 Brashears, M.M.; Galyean, M.L.; Loneragan, G.H.; Mann, J.E.; Killinger Mann, K Prevalence of Escherichia coli O157:H7 and performance by beef feedlot cattle given Lactobacillus direct fed microbials J Food Prot 2003, 66 (5), 748 754 Ghorbani, G.R.; Morgavi, D.P.; Beauchemin, K.A.; Leedle, J.A.Z Effects of bacterial direct fed microbials on ruminal fermentation, blood variables, and the micro bial populations of feedlot cattle J Anim Sci 2002, 80 (7), 1977 1985 Nocek, J.E.; Kautz, W.P.; Leedle, J.A.Z.; Block, E Direct fed microbial supplementation on the performance of dairy cattle during the transition period J Dairy Sci 2003, 86 (1), 331 335 Gilliland, S.E Enumeration and identification of lactoba cilli in feed supplements marketed as sources of Lactoba cillus acidophilus Animal Science Research Report Okla Agric Exp Sta MP 1981, 108, 192 193 Gilliland, S.E Health and nutritional benefits from lactic acid bacteria FEMS Microbiol Rev 1990, 87 (1), 175 188 Krehbiel, C.R.; Rust, S.R.; Zhang, G.; Gilliland, S.E Bacterial direct fed microbials in ruminant diets; perform ance response and mode of action J Anim Sci 2003, 81 (E Suppl 2), E120 E132 Pascual, M.; Hugas, M.; Badiola, J.I.; Monfort, J.M.; Garriga, M Lactobacillus salivarius CTC2197 prevents Salmonella enteritidis colonization in chickens Appl Environ Microbiol 1999, 65 (11), 4981 4986 Haddadin, M.S.Y.; Abdubralim, S.M.; Hashlamoun, E.A.R.; Robinson, R.K The effect of Lactobacillus acidophilus on the production and chemical composition of hen’s eggs Poultry Sci 2003, 75 (4), 491 494 Cromwell, S Antimicrobial and Promicrobial Agents In Swine Nutrition, 2nd Ed.; CRC Press: Boca Raton, FL, 2001; 401 426 Abe, F.; Ishibashi, N.; Shimamura, S Effect of adminis 10 11 12 13 Proteins Guoyao Wu Jon Tate Self Texas A&M University, College Station, Texas, U.S.A INTRODUCTION Proteins are macromolecules consisting of one or more polypeptide chains synthesized from amino acids A peptide may contain approximately 40 to more than 400 aamino acids The word protein originated from the Greek ‘‘proteios,’’ meaning prime or primary This is very appropriate, since proteins are the most fundamental component of animal tissues Proteins play important roles in the body, including their roles in enzyme-catalyzed reactions, muscle contraction, hormone-mediated effects, cell structure, immune response, oxygen storage and transport, nutrition, metabolic regulation, and gene expression The balance between protein synthesis and degradation determines whether a tissue grows or atrophies Thus, knowledge of protein biochemistry and nutrition is of enormous importance for both animal agriculture and medicine bility of proteins to heat damage is increased in the presence of carbohydrates, owing to the Maillard reaction, which involves a condensation between the carbonyl group of a reducing sugar with the free amino group of an amino acid residue (e.g., lysine) Crude protein content in animal tissues and feeds is often obtained by multiplying the nitrogen content by a factor of 6.25, on the basis of the average nitrogen content (16%) in protein Such calculation, however, is not very precise, because some proteins contain less or more nitrogen and because some nitrogenous compounds (e.g., ammonia, urea, amides, choline, betaine, purines, pyrimidines, nitrite, and nitrate) are neither proteins nor amino acids The composition of amino acids in protein is often determined using liquid or gas chromatography PROTEIN NUTRITION AND METABOLISM PROTEIN STRUCTURE AND PROPERTIES Protein Digestion and Absorption Amino acid residues in protein are linked by peptide bonds ( CO NH ) There are four orders of protein structure:[1] primary structure (the sequence of amino acids along the polypeptide chain); secondary structure (the conformation of the polypeptide backbone); tertiary structure (the three-dimensional arrangement of protein); and quaternary structure (the spatial arrangement of polypeptide subunits) The forces stabilizing polypeptide aggregates are hydrogen and electrostatic bonds between amino acid residues Proteins can be classified according to their overall shape (globular or fibrous), solubility in water (hydrophobic or hydrophilic), three-dimensional structure, or biologic function (Table 1) For example, albumin and hemoglobin are globular proteins Fibrous proteins include collagens, elastin, a-keratins (wool and hair), and bkeratins (the feathers, skin, beaks, and scales of most birds and reptiles) Collagens are rich in proline and glycine (approximately 1/3 each), and constitute approximately 30% of total proteins in animals Keratins are rich in cysteine; wool protein contains approximately 4% sulfur All proteins can be denatured by heat, acids, bases, alcohols, urea, and salts of heavy metals The suscepti- Except for the absorption of intact immunoglobulins by the small intestine of mammalian neonates, dietary proteins have no nutritional values until they are hydrolyzed to short-chain peptides and free amino acids in the digestive tract In nonruminants, the digestion starts in the stomach (pH= approximately 3), where protein is denatured by hydrochloric acid, followed by digestion with proteases (pepsins A, B, and C, and renin) The resulting large peptides enter the small intestine to be further hydrolyzed by proteases (including trypsin, chymotrypsin, elastase, carboxyl peptidases, and aminopeptidases) in an alkaline medium (owing to bile salts, pancreatic juice, and duodenal secretions) These enzymes release small peptides and considerable amounts of free amino acids Oligopeptides composed of more than three amino acid residues are further hydrolyzed extracellularly by peptidases (located mainly on the brush border of enterocytes, and to a lesser extent, in the intestinal lumen) to form tripeptides, dipeptides, and free amino acids Major mechanisms for the intestinal absorption of amino acids include both Na+-dependent and Na+independent systems Dipeptides and tripeptides are absorbed intact into enterocytes of the small intestine Encyclopedia of Animal Science DOI: 10.1081/E EAS 120019779 Copyright D 2005 by Marcel Dekker, Inc All rights reserved 757 758 Proteins Table Roles of proteins in animals Roles Muscle contraction Enzyme catalyzed reactions Gene expression Hormone mediated effects Protection Regulation Storage of nutrients and O2 Cell structure Transport of nutrients and O2 Examples of proteins Actin, myosin, tubulin Dehydrogenase, kinase, synthase DNA binding proteins, histones, repressor proteins Insulin, somatotropin, placental lactogen Blood clotting factors, immunoglobulins, interferon Calmodulin, leptin, osteopontin Ferritin, metallothionein, myoglobin Collagen, elastin, proteoglycans Albumin, hemoglobin, plasma lipoproteins through H+-gradient-driven peptide transporters Once inside enterocytes, peptides are hydrolyzed by peptidases to form free amino acids The small intestine transports short-chain peptides (2 amino acid residues) at a faster rate than free amino acids In ruminants, dietary protein is hydrolyzed by ruminal microbial proteases to form small peptides and free amino acids.[2] Amino acids are further degraded to form ammonia, short-chain fatty acids, and CO2 Small peptides, amino acids, and ammonia are utilized by microorganisms in the presence of adequate energy supply (carbohydrates) to synthesize new amino acids, protein, nucleic acids, and other nitrogenous substances The most important initial reaction for microbial ammonia assimilation is catalyzed by glutamate dehydrogenase to produce glutamate, which is then utilized to synthesize glutamine, alanine, aspartate, and asparagine by glutamine synthetase, glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, and asparagine synthetase, respectively These amino acids serve as substrates for the synthesis of all other amino acids by microorganisms in the presence of sulfur and adenosine 5’-triphosphate (ATP) Ruminal protozoa cannot utilize ammonia, but derive their nitrogen by engulfing bacteria and digesting them with powerful intracellular proteases Ammonia that cannot be fixed by ruminal microorganisms is absorbed into blood for conversion into urea via the hepatic urea cycle and may be utilized by ruminal epithelial cells for biosynthetic processes Microbial cells (bacteria and protozoa) containing proteins and amino acids, as well as undigested dietary proteins, leave the reticulorumen and omasum, and enter the abomasum and small intestine, where digestion of protein is similar to that in nonruminants Protein Metabolism In both nonruminants and ruminants, there is extensive first-pass intestinal catabolism and/or utilization of the amino acids absorbed from the lumen of the small intestine, which substantially reduces their availability to extraintestinal tissues and selectively alters the patterns of amino acids in the portal vein.[3] Amino acids that enter systemic circulation may be oxidized to provide ATP and/ or utilized to synthesize glucose, ketone bodies, protein, urea, uric acid, and other nitrogenous substances Dietary protein and energy intake regulate intracellular protein synthesis and degradation (protein turnover) At least four and two ATP molecules, respectively, are required to incorporate one amino acid into a peptide and to hydrolyze one peptide bond Intracellular protein turnover accounts for approximately 15% and 20% of total energy expenditure in adult and growing animals, respectively Whereas protein synthesis is well-characterized, the pathways for intracellular protein degradation are less understood.[1] Lysosomal proteases and cytosolic calpains (Ca2 +-dependent proteases) contribute substantially to the degradation of long-lived, endocytosed, and myofibrillar proteins Proteasome (a multisubunit protease complex) selectively degrades intracellular proteins via the ubiquitination pathway Protein half-lives, which range from < 30 for ornithine decarboxylase to > 50 200 h for lactate dehydrogenase, are determined by N-terminal residue and physicochemical properties of a given protein Protein Requirements Proportions of dietary amino acids have a profound impact on the food intake, growth, and health of animals A limiting amino acid (one that is in the shortest supply from the diet relative to its requirement by animals) impairs the utilization of dietary protein Likewise, an amino acid imbalance (disproportions of dietary amino acids) reduces the feed intake and growth of animals Amino acid imbalances may occur among amino acids regardless of their structure and can be prevented by addition of one or more of the limiting amino acids to the diet Also, an amino acid antagonism (growth depression caused by an excessive intake of an amino acid) commonly occurs among structurally related amino acids (e.g., lysine-arginine, leucine-isoleucine-valine, and threonine-tryptophan) but can be overcome by addition of a structurally similar amino acid Thus, determining optimal amino acid patterns in the diet is very beneficial Nitrogen balance studies and growth trials have long been used to determine amino acid and protein requirements of animals.[4] Minimal requirements can also be Proteins 759 Table Nutritionally essential and nonessential amino acids in monogastric animals Monogastric mammals EAA Poultry NEAA a Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine EAA NEAA Alanine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Prolineb Serine Tyrosine Arginine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Threonine Tryptophan Valine Alanine Asparagine Aspartate Cysteine Glutamate Glutamine Serine Tyrosine a Arginine may not be required in the diet to maintain nitrogen balance in most adult mammals but its deficiency in the diet may result in metabolic, neurological, or reproductive disorders b Proline is an essential amino acid for young pigs estimated by factorial analysis; namely, the sum of fecal and urinary nitrogen in response to a protein-free diet (maintenance), nitrogen deposited in the body, and nitrogen excreted as animal products (e.g., milk, egg, wool, fetus growth) Most recently, direct and indirect (indicator) amino acid oxidation techniques involving radioisotopes or stable isotopes have been developed to estimate requirements of protein and essential amino acids by animals Amino acids are traditionally classified as nutritionally essential (indispensable) or nonessential (dispensable), on the basis of whether they need to be supplied in the diet to maintain nitrogen balance or support the maximal growth of animals (Table 2) Essential amino acids are defined as either those amino acids whose carbon skeletons cannot be synthesized by animals or those that are inadequately synthesized in animals relative to needs, and which must be provided from the diet to meet requirements for maintenance, growth, and reproduction Conditionally essential amino acids are those that normally can be synthesized in adequate amounts by animals, but which must be provided from the diet under conditions where rates of utilization are increased relative to rates of synthesis Nonessential amino acids are the amino acids whose carbon skeletons can be synthesized in adequate amounts by animals to meet requirements Collectively, an ideal protein in the diet would consist of an optimal pattern among essential amino acids that corresponds to an animal’s needs Thus, ideal proteins would likely vary with nutritional and physiological needs, including maintenance, protein accretion, egg and wool production, reproduction, and lactation Because of extensive catabolism of amino acids by the small intestine, the pattern among amino acids in animal tissues or products is not necessarily similar to that in the diet Thanks to Baker’s seminal work,[5] the concept of ideal protein has gained acceptance for formulating swine and poultry diets in the United States and worldwide CONCLUSION A major objective of animal agriculture is to produce high-quality protein products, including meats, eggs, wool, and milk An optimal pattern among amino acids in the diet is crucial for maximizing an animal’s growth and production potential while reducing the excretion of fecal and urinary nitrogenous wastes as a source of environmental pollution Because protein is both the most expensive ingredient in diets and also a major component of cells, knowledge about protein metabolism is essential for improving the efficiency of its utilization by animals ACKNOWLEDGMENT Work in our laboratory is supported in part by grants from the U.S Department of Agriculture/National Research Initiative (USDA/NRI) and Texas A&M University REFERENCES Voet, D.; Voet, J.G Biochemistry; John Wiley & Sons, Inc.: New York, NY, 1995 Stevens, C.E.; Hume, I.D Contributions of microbes in vertebrate gastrointestinal tract to production and conserva tion of nutrients Physiol Rev 1998, 78, 393 427 Wu, G Intestinal mucosal amino acid catabolism J Nutr 1998, 128, 1249 1252 Reeds, P.J.; Hutchens, T.W Protein requirements: From nitrogen balance to functional impact J Nutr 1994, 124, 1754S 1764S Baker, D.H Ideal amino acid profiles for swine and poultry and their applications in feed formulation Biokyowa Tech Rev 1997, 9, 24 ... Spain INTRODUCTION Phytases are meso-inositol hexaphosphate phosphohydrolases that catalyze the initiation of the stepwise phosphate splitting of phytic acid or phytate to lower inositol phosphate... distribution, and appropriate handling and preparation for consumption Proper application of control processes yields products that should be safe for consumption following proper cooking and serving... all species, 60 80% of phosphorus in feeds of plant origin is in the form of phytate (myo-inositol hexakisphosphate) that is poorly available to simple-stomached animals such as swine, poultry,