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Dynamics of Protein Metabolism in the Ruminant.

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Presentation on theme: "Dynamics of Protein Metabolism in the Ruminant."— Presentation transcript:

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2 Dynamics of Protein Metabolism in the Ruminant

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13 Analysis of Dietary Protein Crude protein (CP %) = total N (%)  6.25 Factor is based on 16% N in protein. True protein varies between 13 to 19% N. Source%N in proteinConversion factor oilseed proteins cereal proteins meat or fish alfalfa true microbial protein Not all N in protein is present as true protein.

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17 Classification of protein and nitrogen fractions in feedstuffs Total Acid Detergent Neutral Detergent Borate Buffer Sol A B1 Insol B2 B3 C Sol A1 B1 B2 Insol B3 C Sol A1 B1 B2 B3 Insol C

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19 Crude Protein Non-protein nitrogen True protein (60 to 80%) Essential amino acids Arginine (Arg) Histidine (His) Isoleucine (Ile) Leucine (Leu) Lysine (Lys) Methionine (Met) Phenylalanine (Phe) Threonine (Thr) Tryptophan (Trp) Valine (Val) Non-essential amino acids Alanine (Ala) Asparagine (Asn) Aspartic acid (Asp) Cysteine (Cys) Glutamic acid (Glu) Glutamine (Gln) Glycine (Gly) Proline (Pro) Serine (Ser) Tyrosine (Tyr) Amides Amines Amino acids Peptides Nucleic acids Nitrates Ammonia Urea Lignified nitrogen

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21 Calculations Hours Calculate slope (change per hour) of each line. Slope = k d, has units of % of pool remaining that is lost per hour. Log of % nutrient remaining Protein Fraction A, B 1 B 2 B 3 C

22 Terms for describing nitrogen components of feedstuffs Degradable Intake Protein (DIP): dietary crude protein degraded in the rumen. Undegraded intake protein (UIP): dietary crude protein that is not degraded in the rumen and escapes or bypasses the rumen to the intestine. It is largely true protein but also contains ADFIP. Soluble protein (SolP): Contains non-protein nitrogen, amino acids and peptides. Soluble protein is degraded instantaneously in the rumen.

23 Terms for describing nitrogen components of feedstuffs Non-protein nitrogen (NPN): Includes amides, amines, amino acids, some peptides, nucleic acids, nitrates, urea, ammonia. Degraded instantaneously in the rumen. Acid detergent fiber insoluble protein (ADFIP): Consists of heat damaged protein and nitrogen associated with lignin. Resists ruminal fermentation and is indigestible in the small intestine.

24 Protein content of common feedstuffs CP DIP UIPSolPNPNADFIP Feedstuff%DM%CP%CP%CP %SolP %CP Alfalfa silage Barley silage Corn silage Alfalfa hay Timothy hay Barley straw Barley grain

25 Protein content of protein supplements CP DIP UIPSolPNPNADFIP Plant sources%DM%CP%CP%CP %SolP %CP Canola meal Soybean meal Soypass  * Brewer’s grains Corn distiller’s gr Corn gluten meal *Commercial product: LignoTech USA, Inc.

26 Protein content of protein supplements CP DIP UIPSolPNPNADFIP %DM%CP%CP%CP %SolP %CP Animal sources Blood meal Feather meal Fishmeal Meat and bone Non-protein nitrogen sources Urea

27 Ruminally Protected Protein A nutrient(s) fed in such a form that provides an increase in the flow of that nutrient(s), unchanged, to the abomasum, yet is available to the animal in the intestine Methods to decrease the rate and extent of ruminal degradation involved the use of heat, chemical agents, or combination of both

28 Heat Processing Heat processing decrease rumen protein degradation by denaturation of proteins and by the formation of protein-CHO (Millard reactions) and protein cross-links. Commercial methods that rely solely on heat include: cooker-expeller, roasting, extrusion, pressure toasting, and micronization. Heat processing reduced fraction A, increases fraction B, and C, and decreases in the fractional rates of degradation of the fraction B

29 Heat Processing cont. Over heating also causes significant losses of lysine, cysine, and arginine. Among those AA, lysine is the most sensitive to heat damage and undergoes both destruction and decreased availability

30 Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteins

31 Heat Processing Careful control of heating conditions is required to optimize the content of digestible RUP. Under heating results in only small increase in digestible RUP.. Over heating reduces the intestinal digestibility of RUP through the formation of indigestible Millard products and protein complexes.

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33 Chemical Treatment Chemical treatment of feed proteins can be divided into three categories: 1) chemicals that combine with and introduce cross-links in proteins, (2) chemicals that alter protein structure by denaturation (e.g., acids, alkalis, and ethanol), and (3) chemicals that bind to proteins but with little or no alteration of protein structure (e.g., tannins).

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35 Chemical Treatment cont. For a variety of reasons, often including less than desired levels of effectiveness, use of chemical agents as the sole treatment for increasing the RUP content of feed proteins has not received commercial acceptance. A more effective approach involving “chemical” agents has been to combine chemical and heat treatments. An example of this approach is the addition of lignosulfonate, a byproduct of the wool pulp industry that contains a variety of sugars (mainly xylose), to oilseed meals before heat treatment.

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37 Chemical Treatment cont. The combined treatments enhance non- enzymatic browning (Millard reactions) because of the enhanced availability of sugar aldehydes that can react with protein.

38 Characterization of Protein Sources Common protein supplements that are high in RUP are: Common protein supplements that are high in RUP are: Fish meal Meat and bone meal (MBM) Feather meal (FtM) Blood meal (BM) Corn gluten meal (CGM) Distillers dried grains (DDG) DDG with solubles (DDGS) Brewers dried grains (BDG) Brewers wet grains (BWG)

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48 Nitrogen transactions in the rumen Sources of nitrogen in the rumen Dietary crude protein (true protein and NPN). Recycled microbial protein (bacteria and protozoa). Endogenous N (urea, abraded epithelial cells, salivary proteins).

49 Degradation of nitrogenous compounds by ruminal microorganisms Bacteria 30 to 50% of the bacteria are proteolytic. Most species have some activity with the exception of the main cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus flavefacians, R. albus). Major proteolytic bacteria: Ruminobacter amylophilus, Butyrivibrio Fibrisolvens and Prevotella ruminicola. P. ruminicola is the most numerous proteolytic bacteria (> 60% of ruminal bacteria) with strains that occur on both roughage and mixed roughage- concentrate diets.

50 Bacteria cont’d R. amylophilus is the most active proteolytic bacteria. Important on starch-based diets. Breakdown of both soluble and insoluble protein in the rumen. Protozoa Minor involvement in soluble protein breakdown. Engulf and hydrolyze particulate proteins and bacteria. Predatory activity of protozoa against rumen bacteria contributes to bacterial protein degradation and turnover in the rumen. Fungi Minor role in protein degradation.

51 PROTEIN OLIGOPEPTIDES DIPEPTIDES AMINO ACIDS AMMONIA D. ruminantium, B. fibrisolvens, E. caudatum Clostridium spp, E. simplex, E. budayi E. caudatum ecaudatum, E. ruminantium, E. maggii Fusobacterium spp., E. medium L. multipara O. caudatus, P. ruminicola P. multivesiculatum, R. amylophilus, S. ruminantium O. joyonii, N. frontalis, S. bovis, P. communis S. bovis, R. amylophilus, P. ruminicola D. ruminantium, E. caudatum F. succinogenes, M. elsdenii, P. ruminicola Isotricha spp., L. multipara, S. ruminantium C. aminophilum, C. sticklandii P. anerobius, B. fibrisolvens, P. ruminicola M. elsdenii, S. ruminantium, E. caudatum Isotricha spp. Dipeptidyl peptidase Dipeptidase

52 Properties of ammonia producing bacteria High Numbers Low Activity Butyrivibrio fibrisolvens Megasphaera elsdenii Prevotella ruminicola Selenomonas ruminantium Streptococcus bovis > 10 9 per ml 10 to 20 nmol NH 3 min -1 (mg protein) Low Numbers High Activity Clostridium aminophilum Clostridium sticklandii Peptostreptococcus anaerobius 10 7 per ml 300 nmol NH 3 min -1 (mg protein)

53 Breakdown of NPN in the rumen Major sources of NPN include: dietary NPN, and recycled urea. Extremely rapid and releases ammonia. Major end product of protein degradation in the rumen Ammonia

54 Influence of diet on proteolysis Concentrate Increase in total microbial population, including several of the more active protein degrading bacteria which are also amylolytic (Prevotella rumincola, Ruminobacter amylophilus and Streptococcus bovis). Fresh forage Increase in the proportion of proteolytic bacteria relative to total microbial population.

55 Microbial protein synthesis in the rumen

56 Factors Influencing Microbial Protein Synthesis Ammonia Most important source of N for bacterial protein synthesis. 50 to 80% of bacterial N is derived from ammonia. Bacteria hydrolyzing structural carbohydrates utilize ammonia as N source. Several mechanisms for the uptake of ammonia:  high affinity, low Km (ammonia concentration) enzyme system glutamate synthetase - glutamate synthase (GS-GOGAT)  lower affinity, higher Km system NADP-glutamate dehydrogenase (NADP-GDH), NAD-GDH and alanine dehydrogenase. Minimum level of ammonia is necessary for maximum growth and efficiency (5 mg/100 ml of rumen fluid).

57 Peptides and amino acids 20 to 50% of ruminal microbial N is derived from this pool. Supplying preformed peptides and amino acids spares the cost associated with synthesizing amino acids. Rapidly fermenting organisms, bacteria hydrolyzing non-structural carbohydrates (starch, pectin, sugars), utilize peptides, amino acids and ammonia. Availability of peptides improves microbial growth.

58 Synchronization of protein and carbohydrate degradation Microbial protein synthesis is maximized when the release of N from protein occurs with the release of energy from the degradation of carbohydrates. Fractional Outflow Rates Increasing the rate of passage removes the more mature organisms, reducing the median age of the microbes. Reduces the amount of energy expended on maintenance so more energy can be used for growth.

59 Efficiency of Microbial Growth Diet % of TDN (DOM) Rate of passage pH BCP/100 gm TDN

60 Effect of dilution rate on YATP.

61 Reduces the amount of intraruminal N recycling (microbial protein turnover).

62 Intraruminal nitrogen recycling Turnover of bacteria and protozoa. 30 to 55% of bacterial N 75 to 90% of protozoal N Causes of microbial N recycling Engulfment and subsequent digestion of bacterial cells by protozoa Lysis due to autolytic enzymes, bacteriocins, or other soluble compounds in response to nutrient deprivation or interspecies competition Activity of bacteriophages and mycoplasmas.

63 Ammonia accumulation in the rumen Ammonia concentration exceeds the capacity of the ruminal bacteria to utilize it. Absorbed across the ruminal wall into the blood where it is transported to the liver and metabolized to urea. Urea is filtered by the kidney and excreted in urine as waste N. In addition to poor N retention, the synthesis of urea from ammonia also has an energetic cost (12 kcal/g N) to the animal.

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70 Urea recycling Blood urea originates from the endogenous metabolism of tissue protein, the deamination of excess absorbed amino acids and the absorption of ruminal ammonia. Recycled to the rumen primarily through the rumen wall and to a lesser extent via saliva (approx 15% of urea recycled to the rumen is via saliva) Facultative microorganisms located on the rumen epithelium wall have urease activity

71 Factors involved in increasing the permeability of the rumen wall to urea

72 Composition of microbial protein reaching the intestine Hay Hay and Conc sheep 1 sheep 2 sheep 1 sheep2 N in rumen digesta, g Fungi Protozoa Bacteria N in rumen digesta, % of total microbial N Fungi Protozoa Bacteria N in duodenal digesta, g Fungi Protozoa Bacteria N in duodenal digesta, % of total microbial N flow Fungi Protozoa Bacteria

73 Undegraded dietary protein Protein that escapes microbial degradation passes to the lower digestive tract where it will be largely degraded. Only the very refractive N component such as N bound to lignin or products of the Maillard reaction will not be degraded. Benefit to the animal of supplying UIP will depend on the provision of essential amino acids that are required in excess of what is supplied by microbial protein.

74 Protein digestion in the abomasum Pepsinogen (inactive) Pepsin (hydrolysis bonds at carboxylic end of aromatic AA and Leu) HCl Protein Denatured protein disruption of non-covalent bonds uncoiling of protein HCl Denatured protein Small polypeptides few amino acid Pepsin pH 1.6 to 3.2

75 Secretion and activation of pancreatic and intestinal proteolytic enzymes Polypeptides Short peptides AA Intestinal endocrine cell Pancreatic acinar cell Intestinal mucosal cell Trypsinogen Trypsin Chymotrypsinogen Proelastase Procarboxypeptidase Cholecystokinin (CCK) CCK and Secretin Enterokinase Chymotrypsin Elastase Carboxypeptidase

76 Sites of hydrolysis of proteolytic enzymes Pancreas TrypsinDibasic AA (Arg, Lys), C-terminal end ChymotrypsinAromatic C terminal peptides ElastaseNeutral C terminal peptides CarboxypeptidaseC-terminal end Intestine EnteropeptidaseN-terminal end

77 Digestion in the small intestine Polypeptides Oligopeptides Amino acids Dipeptides Tripeptides Pancreatic and intestinal proteases Dipeptides Tripeptides Amino acids Intestinal di- and tripeptidases (cell membrane and cytosol)

78 Protein absorption Small intestine Major site of absorption Amino acids absorbed in the ileum Dipeptides and tripeptides absorbed in the jejunum Active transport (energy dependent) 9 carrier systems for amino acids specific for certain amino acids

79 Protein metabolism Intestinal cell  Glu, Asp, Gln metabolized by intestinal cell  provides 40% of energy requirements Liver  protein synthesis  synthesis of non-essential amino acids  C-skeletons catabolized for energy and the amine group metabolized to urea

80 Nitrogen metabolism in the large intestine N supplied to the lower tract comes from the recycling of urea and other endogenous protein (sloughed epithelial cells, enzymes and glycoproteins of mucus). Energy substrates come from the residual fermentable fibre, the glycocalyx of rumen microorganisms, starch and other polysaccharides that have resisted rumen and enteric digestion. As the amount of fermentable energy from the diet reaching the lower tract increases, microbial synthesis increases and fecal N excretion increases.

81 Routes of nitrogen excretion Urine (urea) Endogenous urinary N from the catabolism of tissue proteins Absorption and metabolism of excess ruminal ammonia. Catabolism of excess absorbed amino acids Feces Microbial N synthesized in and passed from the large intestine. Sloughed cells and secretions of the GI tract. Undigested unabsorbed dietary protein.

82 NPN Indigestible Undegradable in rumen Degradable in rumen Peptides amino acids Microbial N Peptides amino acids Microbial N Feces Plasma urea Tissues Maintenance Growth Conceptus Lactation Wool Urine Digestible Feed NH 3 Endog N NH 3 Rumen Small intestine Large intestine Energy

83 Meeting the protein requirements of ruminant animals Degradable intake protein in the rumen for ruminal microorganisms to maximize digestibility of the diet and feed intake. Absorbable essential amino acids at the intestine from the digestion of microbial protein produced in the rumen and dietary intake protein that escapes rumen fermentation.

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88 Defaunation (protozoal removal) Removal of protozoal predation of bacteria. Increases substrates (starch) available for fermentation and growth by bacteria. Increases amount of bacterial protein synthesized in the rumen. Increases the flow of microbial protein from the rumen. Reduction in ammonia concentration.

89 Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteins

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