Comparative Animal Nutrition and Metabolism Peter R. Cheeke Professor Emeritus Department of Animal Sciences Oregon State University, USA and Ellen S. Dierenfeld Novus International St Louis, Missouri, USAIntroduction Nutrition is a very broad discipline, encompassing. various aspects of biochemistry, physiology, endo crinology, immunology, microbiology and pathol- ogy. Discussion of virtually any aspect of nutrition draws upon some prior knowledge of these dise plines. Nutrition can be defined as applied bio- chemistry, and is commonly referred to as nutritional science. Animal nutrition has conventionally meant the study of nutritional needs of domestic animals, in contrast to human nutrition, which specifically targets humans. Dietetics refers to the formulation and preparation of diets to meet the needs of humans. The comparable activity with domestic animals is diet formulation. Nutritional science deals with the principles of nutrition, while dietetic and diet formulation deal with their application. Animal and poultry nutritionists have played key roles in the development of the nutritional sciences. Many of the nutrients were discovered by animal nutritionists using chicks or rats as experimental animals, Human nutrition developed more slowly as a scientific discipline, emerging in most cases from the discipline of Home Economics. The objectives of this book are to clearly explain principles of animal nutrition, A compar approach is taken, recognizing that there are con- siderable differences in nutrient digestion, metabo- lism and requirements among various mammalian and avian species. On a molecular level, the sim larities in metabolic processes among animals are far greater than the differences, reflecting their common evolutionary history. However, the pas- sage of nutrients from the environment to the molecular level of the animal cell involves great species differences, largely because of differences in food selection and food-sceking strategy, and in digestive tract physiology and digestive strategies. These comparative differences will be explained and their significance to nutritional needs dis- cussed. Within categories of animals, there are similarities in the nutrition of domestic and wild species. For example, there ate similarities among wild avian species (i. birds) to domestic poultry, as well as numerous differences. Digestive pro- cesses in hindgut digester wild animals, such as elephants, share some similarities with domestic species such as horses, whereas the koala resembles, the rabbit in hindgut function, The basic nutri tional requirements and digestive physiology of wild ruminants such as deer, antelope and giraffes resemble to some extent those of domestic rumi- nants such as cattle, sheep and goats. Its intended that this text will be relevant and useful to those interested in wildlife nutrition as well as to those primarily interested in domestic animals. The prin- ciples involved are similar. While it is true that more nutritional research has been conducted with domestic livestock species than with wild animals, animal scientists are perhaps not generally appre~ ciative of the degree to which findings wi domestic species can influence the understanding of nutritional principles. For example, a greater understanding of the variations in rumen anatomy and physiology of wild ruminants could lead to greater recognition by animal scientists that it is simplistic to view sheep, goats and cattle as func~ tionally equivalent in terms of digestive functions and processes. Thus, itis anticipated that the com- parative approach developed in this text wil enhance the appreciation of nutritional principles by students having either domestic a life orientations, non ial or wild- The Nutrients Nutrients may be defined as dietary essentials for one or more species of animal, implying that not all animals require all nutrients. For example, few species besides primates and the guinea pig have a dietary requirement for vitamin C. Ruminant animals do not normally have a dietary requite- ment for B-complex vitamins and amino acids.Other nutrients can in fact be omitted from the diet if appropriate dietary adjustments are made. There is no dietary requirement for carbohydrates per se, either individually or collectively. While glucose is an essential metabolite in mammalian metabolism, it is not a dietary essential. Nevertheless, glucose and other sugars are commonly considered to be nutrients. This ambiguity about what is and what is not a nutrient exists primarily with energy- yielding substances, for which there is not a specific requirement for individual sugars and fatty acids, but rather there is a collective requirement for car- bohydrates, fats and amino acids which can be metabolized to provide energy. For minerals and vitamins, the requirements are unequivocal; they have specific metabolic roles which cannot be replaced by other nutrients, Those substances that can be considered nutri- ents fit into one of the following six catego proteins; carbohydrates; lipids; minerals; vitamins; and water. Water is considered to be a nutrient, although for domestic animals it does not totally fit the defini- mn of a nutrient because it is not generally required in the diet (food) but is usually consumed separately as drinking water. Some desert animals (e.g. pack rat) never drink, but survive on meta- bolic water (see Chapter 20). Marine mammals (e.g. seals, sea lions) never drink, but obtain their water from their diet (fish tissue), while fruit-eating animals (frugivores) obtain a major portion of their water from the diet (fruit). Thus water is truly a nutrient for these species. A brief description of each of these major nutri- ent categories will be given for introductory pur- poses; they will be discussed in detail in later chapters. Proteins Proteins are large molecules composed of amino acids joined together by peptide bonds (see Chapter 4). Plant and animal proteins ate composed of about 20 amino acids, arranged in various sequences to form specific proteins. A few other amino acids (c.g. citrulline) do not occur in protein tissue but have other specific functions, and are known as non-protein amino acids, Over 300 individual amino acids are known; most of them are non- protein amino acids in plant tissue, with no role in animal nutrition (except a negative one if they are toxic), Some sources indicate even greater numbers of known amino acids in plants, as high as over 900 (Wink, 1997). The genetic control of protein syn- thesis involving DNA and RNA metabolism is one of the marvels of life. Each cell (except non-avian erythrocytes) contains a genetic code, programming the cell to synthesize particular proteins. Proteins ae an integral part of animal structure and meta- olism. They constitute a major part of the body structure, as components of muscle, connective tis- sue and cell membranes. All metabolic reactions are dependent on proteinaceous enzymes. A major concern of animal nutritionists is the pro- vision of adequate dietary protein and amino acids. From a comparative standpoint, there are great dif- ferences in protein utilization, with ruminant animals largely insulated from specific dietary amino acid requirements because of the activities of rumen microbes, whereas carnivores have some distinet dif- ferences from omnivores in amino acid needs, All amino acids contain nitrogen; therefore, all proteins contain nitrogen. Protein utilization by animals is often studied by measuring nitrogen in nitrogen balance trials. Protein digestibility, for example, is determined by measuring the nitrogen content of feed and faeces to determine the amount of absorbed (hence digested) nitrogen, reflective of amino acid absorption. In general, proteins contain about 16% nitrogen. The protein content of feeds is usually measured by determining the nitrogen content and multiplying it by the factor of 6. Crude protein is defined as N 6.25 (16 of nitro- gen (N) come from 100g protein; therefore, 1g of nitrogen is associated with 100/16 = 6.25 g of pro- tein). Nitrogen is measured by the Kjeldahl proc dure, which is named after the Danish chemist who developed it. The feed sample is boiled in concen: trated sulfuric acid, resulting in the complete oxi- dation of all organic material. The proteins and amino acids are completely degraded; their nitro- xen is released as ammonium ion (NH). The solu- tion is then made alkaline, converting NH to ammonia (NH,). Steam is passed through the solu- tion (steam distillation), driving off the NH,, which is trapped in a boric acid solution, The concentra~ tion of NH, is measured by titration. It is impor tant to recognize that the erude protein procedure measures nitrogen. Thus, it does not distinguishnecessary for the synthesis of steroid hormones and bil acids and is an essential component of cell membranes. In plants, other important lipids include the photosynthetic pigments (chlorophyll and carotenoids), and waxes and cutin which form the water-shedding waxy surface of leaves. Minerals Mineral elements ate the inorganic components of plant and animal tissue. In animal nutrition, they are classified in two categories: (i) macro-minerals; and (ii) micro (trace) minerals (elements). The macro-clements are required in relatively large quantities; trace elements are required in very small amounts. The macro-elements calcium, phospho- rus and magnesium are major components of the skeletal system, and thus function in a structural role. The other minerals function primarily in regu- latory roles. For example, sodium, potassium and chlorine are involved as electrolytes in regulating fluid balance between the gut, blood, cells, tissue spaces and body cavities. Sulfur as such is not a dietary essential, but is a component of many organic constituents of tissues such as the sulfur- containing amino acids, the vitamins biotin and thiamin, mucopolysaccharides such as chondroitin sulfate, and the metabolically essential coenzyme A (CoA). Other elements regulate enzyme activity, either as integral components of enzymes or as cofactors. Examples include selenium as an integral component of the enzyme glutathione peroxidase and copper as a cofactor of cytochrome oxidase. The following are the generally recognized nutri- tionally essential elements: © Macro-element: calcium, phosphorus, sodium, potassium, chlorine, magnesium, sulfur. © Trace elements ~ manganese, zinc, iron, copper molybdenum, selenium, iodine, cobalt, chromium, There are a few other minerals, such as vanadium, silicon, tin, boron and nickel, for which dietary need has been demonstrated only with laboratory animals fed highly purified diets. They are unlikely to be of practical importance for animals consuming diets composed of naturally occurring constituents. Vitamins, The term ‘vitamin’ was coined in 1912 by a Polish chemist, Casimir Funk, who discovered that the antipolyneusits factor in rice husks was nitrogenous in nature. He called it a ‘vital amine’, and proposed the term ‘vitamine’ for accessory food factors. Vitamins can, like minerals, also be classified into two groups: (i) the fat-soluble; and (ji) water-soluble vitamins. The fat-soluble vitamins are vitamin A (retinol), vitamin D (cholecalciferol), vitamin E (a-tocopherol) and vitamin K (phylloquinone). The water-soluble vitamins are vitamin C (ascorbic acid) and the members of the vitamin B-complex group. These include vitamin B, (thiamin), vitamin B, (ribo- flavin), vitamin B, (pyridoxine), vitamin B,, (cyano- cobalamin), niacin (nicotinic acid), folacin (folic acid), biotin, choline and pantothenic acid. It is pref erable to refer to vitamins by their accepted names (e.g. thiamin) rather than by the B-designation (e.g. vitamin B,). Vitamins are organic compounds other than pro- teins, carbohydrates and lipids that have specific roles in metabolism and are required in the diet in very small amounts.? Deficiency causes a specific disease, which is cured or prevented only by restor- ing the vitamin to the diet. In most cases, these roles are in the regulation of enzyme function. In vitamin deficiencies, the loss of particular enzyme activities results in specific deficiency symptoms. Thus in vitamin K deficiency, as an example, impaired blood clotting occurs because of the enzy- matic role of vitamin K in the clotting process. Vitamin A has a role in vision; hence vitamin A deficiency can result in blindness. The dietary requirements for vitamins are very low in quantitative terms. Once the metabolic need has been satisfied, there is no further response from additional quantities. In spite of this, there is a huge commercial industry, based on an unlikely alliance of multinational drug companies that manufacture vitamins and ‘anti-establishment’ con- cerns such as ‘health food’ stores that sell them, involved in megadosing of vitamins by the human population of the USA! (The excess vitamins wind up in wastewater treatment plants.) There are a number of ‘bogus’ vitamins (e.g. vitamin H, vitamin P) that do not exist. In some cases, they are names coined by entrepreneurs sell- ing dietary supplements. In other cases, they were named by scientists who believed they had isolated a new vitamin, but subsequent work revealed it was either a previously named vitamin or it was a substance that did not meet the criteria for vitamin status (e.g. vitamin B,). The B-complex group does not have all the numbers from 1 to 12, because some of them (e.g. vitamin B,) proved to be invalid.The last vitamin to be discovered was vitamin By. in 1948. Before its chemical identification, extracts with the enzyme activity were called the ‘cow manure factor’ and the ‘animal protein factor’. Dietary supplements (nutraceutic ) A number of dietary supplements are often used in animal feeding, particularly in the horse indus- try. The term ‘supplements’ in this context refers to non-nutritive substances, in contrast to the known nutrients. Those with perceived medicinal properties are sometimes called nutraceuticals. ‘Commonly, a number of herbal products are used. Some herbal products, such as yucca, are known to have anti-inflammatory properties. In general, dictary supplements are unproven scientifically, and their use is promoted by aneedotal ‘evidence’. Lack of scientific proof is a reflection of a lack of financial incentive for research support, because most herbal products are not patentable. The problem with ‘anecdotal evidence’ is that it i often due to the placebo effect (Kienzle et al., 2006). The only acceptable proof is a double- blind study (in which neither the investigator nor the subject knows the identity of which subjects are on which treatment). Subjective types of responses, particularly in equine studies, are also a problem. With dressage horses, for example, the responses are the evaluations of the rides, such as unresponsiveness or stiff back, horse ‘too hot’, and so on. The more subjective the criteria, the more likely that the rider's imagination comes into play (Kienzle et al., 2006). Animal Cellular Metabolism Enzymes and hormones Enzymes and hormones have important roles in regulation of metabolism, Some general comments of an introductory nature will be made here, and specific roles of various enzymes and hormones will be discussed later as appropriate. Enzymes are organic catalysts. Catalysts are sub- stances that accelerate chemical reactions. They undergo chemical changes during the reaction, but revert to their original state when the reaction completed, and can be reused. Virtually every chemical reaction that takes place in living tissues requires an enzyme catalyst, With some exceptions (e.g. drug metabolizing enzymes), enzymes are very substrate-specific, having an active site that is very specific in its binding capabilites. All enzymes are proteins. Enzymes can be extracted from tissues and purified, and utilized in research, medicine and for industrial purposes. Enzymes have traditionally been named for the substrate upon which they act, with the addition of the ending ‘ase’. Some of the enzymes encoun tered in nutrition, such as trypsin, chymotrypsin, pepsin and rhodanese, were named before a unify- ing system of nomenclature was developed. The current system includes information on the type of chemical reaction catalysed and cofactors required. For example, oxidoreductases catalyse oxidation-reduction reactions, transferases cata- lyse transfer of a group (e.g. glutathione- ferase), hydrolases act to hydrolyse ester bonds, isomerases catalyse interconversions of isomers (e.g. trans-vitamin A to cis-vitamin A), etc. Essential cofactors, such as nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), are included in the name. The result can be some real tongue-twisters, such as UDP-N-acetylglucos- amine:dolicholphosphate N-acetylglucosamine phosphate transferase! This enzyme is essential for formation of glycoproteins, and is inhibited by the mycotoxins responsible for annual ryegrass toxicity of sheep. Cofactors associate reversibly with enzymes or substrates; they are often metal ions. Enzymes that require a metal ion cofactor are called metal- activated enzymes, in contrast to metalloenzymes that contain a metal ion as a prosthetic group. Copper, iron and zine frequently function as cofac- tors. Organic cofactors are called coenzymes. Coenzymes act as reusable shuttles that can trans- port substrates from their point of generation to their point of utilization. Several B-vitamins func- tion as constituents of coenzymes, including pan- tothenic acid (coenzyme A, CoA), niacin (NAD, NADP), riboflavin (flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD), thiamin, folic acid and vitamin B,,. Many coenzymes are deriva- tives of adenosine monophosphate (c.g. CoA, NAD, NADP, FAD). The distribution of enzymes in living tissues is highly ordered, with enzymes located so that the product(s) of one enzymatic reaction are substrates for the next reaction. They are compartmentalized in subcellular fractions; the enzymes of glycolysis for example, are in the cell eytosol while the citr acid cyele enzymes are in the mitochondria. transEnzymes have an ‘active site’ or ‘catalytic site” where the interactions between enzyme, substrate and coenzymes occur. Many enzymes are produced and secreted as proenzymes or zymogens, which must be activated to the active enzyme form. This process is physio- logically necessary in many cases, such as when the enzyme is needed intermittently, but when it is needed, itis needed immediately. Prothrombin is a proenzyme in the blood; when blood clotting is required, prothrombin is activated to the enzyme thrombin. Pepsinogen is secreted from the gastric mucosa, and activated by hydrochloric acid (HCI) in the stomach to pepsin. Other proteolytic enzymes are secreted as zymogens (e.g. trypsinogen, chymo- trypsinogen). It would be physiologically difficult for a cell to store an active proteolytic enzyme without digesting itself. Many enzymes require metal ions for their activ- ity. Metalloenzymes contain a mineral as an inte- gral part of their structure, while metal-activated ‘enzymes require the presence of a less tightly bound mineral (cofactors). There are many zine and cop- per metalloenzymes, such as thymidine kinase (Zn) and cytochrome oxidase (Cu). The activity of enzymes is regulated in various ys in order to direct metabolic activity to main- tain homeostasis. Homeostasis refers to the con- stancy of the internal environment, whereby cellular metabolism is regulated to attempt to maintain a steady-state condition. An example is blood glucose. Homeostatic hormones such as insulin and glucagon function to maintain a fairly constant blood glucose concentration. Marked deviations from normal are pathological (e.g. dia- betes). Another example is serum calcium, which inmost species is homeostatically maintained within a narrow concentration range. Enzyme con- centration can be increased by inducers. For exam- ple, many drugs and natural toxins are detoxified by drug-metabolizing enzymes (mixed function oxidases) in the liver. Many of these toxins act as inducing agents, inducing increased concentration of the enzymes req! Conversely, enzymes involved in nutrient metabo- lism may be ‘turned off” by feedback inhibition. When adequate quantities of a product have been produced and begin to accumulate, one or more of the enzymes involved in its production may he inhibited. The metabolism of glucose and directing it to either ATP production or energy storage as glycogen is regulated by feedback inhi red to detoxify them. Many enzymes are regulated by hormones such as thyroxine which regulates the metabolism of glu- ‘cose and fat. Hormones, along with enzymes, are intimately involved in animal metabolism. Classically, hormones have been defined as substances that are produced in one tissue (a gland) and transported to a target tissue, with their production or release regulated by positive or negative feedback inhibi- tion. It is now recognized that, in addition to target tissues, hormones can also act in non-target adja- ccent tissues as well as in the cells in which they are produced. Many hormones are nutritionally impor- tant. These include thyroid hormones (thyroxine and calcitonin), pancreatic hormones (insulin and sglucagon), parathyroid hormone, growth hormone, and a number of gastrointestinal hormones. Many hormones are proteinaceous in nature, being . thyroxine and serotonin is derived from tryptophan) or consisting of one or more polypeptide chains (c.g. insulin, glucagon, parathyroid hormone, growth hormone). Others are lipids, derived from cholesterol (e.g. androgens, ‘oestrogens and 1,25 dihydroxycholecalciferol) and fatty acids (e.g. prostaglandins), To exert their physiological effects, hormones react with receptor molecules. These may be within the cell (e.g, thyroid hormones bind to intracellular receptors) of at the cell surface. In the latter case, the cell surface receptor releases a second messen- ‘ger, which mediates the intracellular effects of the hormone. For example, a common second messen- ger is cyclic adenosine monophosphate (cAMP). Hormones and enzymes are thus intimately co- involved in regulating animal metabolism. Receptor activity can be modified to regulate cellular uptake according to need. Activity is up-regulated to increase inflow of a substance, and down-regulated to decrease its uptake. Metabolism and metabolomics Metabolism processes in living tissue. Catabolism refers to the breakdown or oxidation of fuels, while anabolism refers to the synthetic reactions that build up tissues (cg. protein synthesis). The combined catabolic and anabolic processes constitute metabolism. Metabolomics is an emerging discipline, similar in concept to other ‘omies’ such as genomics and pro- . Metabolomics is the ‘big-picture’ study of rs to the summation of biochemicalall metabolites ~ all molecules smaller than pro- teins and polynucleotides - in an individual. This concept recognizes ‘biochemical individuality’, in that each individual has a unique internal chemis- try (the metabolome). Hopefully the ultimate result of the understanding of an individual's metabolome will be a recognition of metabolic “choke points’ and the ability to modify nuttitional status to optimize internal chemistry. The disci- pline is in its infancy. The concept was first advanced in a classic book, Biochemical Individuality, by Roger J. Williams (first published 1956; reprinted 1998), Williams proposed that each individual is biochemically unique, and has unique nutritional requirements. He recognized that nutritional status can influence the expression of genetic characteristics. It is only recently that advances in insteumentation and analytical rech- niques have permitted determination of unique metabolic characteristics (the metabolome). Metabolites (metabolic intermediates) are sub- stances formed during the catabolism or anabolism of nutrients. They are generally small molecules. For example, pyruvic acid is a metabolite of glu- cose; it is an intermediate formed during the catabolism of glucose by a metabolic pathway (gly- colysis). A metabolic pathway is a series of bio- chemical reactions catalysed by enzymes, and generally proceeds from a state of higher energy to lower energy. In 1942, a German biochemist, R. Schoenheimer, published a book entitled The Dynamic State of Body Constituents. By the use of radioactive iso- topes, he had discovered that many tis constant state of catabolism-anabolism, or mobilized and replaced by newly formed tissue. For ‘example, adipose tissue is not an inert deposit of lipid; it is continually being mobilized and replaced. by new lipid (see Chapter 15). Muscle tissue under- ‘goes degradation and re-formation. Humans turn ‘over 1-2% of their total body protein daily. Liver proteins have half-lives of 30min to several hours. Bone is a dynamic structure that undergoes con- tinuing remodelling, with mobilization of bone mineral followed by deposition of new bone tissue (see Chapter 18).