12 Analysis of Dietary Protein Crude protein (CP %) = total N (%) 6.25Factor is based on 16% N in protein.True protein varies between 13 to 19% N.Source %N in protein Conversion factoroilseed proteinscereal proteinsmeat or fishalfalfatrue microbial proteinNot all N in protein is present as true protein.Crude protein provides a rough estimate of dietary protein concentration for two reasons. First it is based on concept that 16% of the weight of protein is N, but the N in true protein varies between 13 to 19%. Secondly, not all N in protein sources is present as true protein.
16 Classification of protein and nitrogen fractions in feedstuffs TotalBorateBufferNeutralDetergentAcidDetergentSolAB1InsolB2B3CSolA1B1B2InsolB3CSolA1B1B2B3InsolCSolubility in borate-phosphate buffer, neutral detergent and acid detergent.Fractions A and B1 are soluble in borate buffer. Precipitation of this fraction with tungstic acid allows the determination of the A fraction. Extraction with neutral detergent buffer isolates fractions A, B1 and B2 (soluble in ND) from B3 and C (insoluble in ND). Acid detergent partitions proteins into fractions C (insoluble in AD), and A and all the B’s (soluble in AD.
18 Crude Protein True protein (60 to 80%) Non-protein nitrogen Lignified Essential amino acidsArginine (Arg)Histidine (His)Isoleucine (Ile)Leucine (Leu)Lysine (Lys)Methionine (Met)Phenylalanine (Phe)Threonine (Thr)Tryptophan (Trp)Valine (Val)Non-essentialamino acidsAlanine (Ala)Asparagine (Asn)Aspartic acid (Asp)Cysteine (Cys)Glutamic acid (Glu)Glutamine (Gln)Glycine (Gly)Proline (Pro)Serine (Ser)Tyrosine (Tyr)AmidesAminesAmino acidsPeptidesNucleic acidsNitratesAmmoniaUreaCrude protein is a useful measure for evaluating diets but it oversimplifies the very complex nitrogenous components of diets.Although some feedstuffs particularly fresh forages and silages contain substantial amounts of organic NPN, from a practical point of view, NPN in formulated feeds refers to added materials, primarily urea.
19 Fraction A contains ammonia, nitrates, amino acids and peptides; ruminal degradation is assumed to be instantaneous and none reaches the small intestine. B1 consists of globulins and some albumins. Because ruminal degradation is 200 to 300 %/h, it is almost completely fermented in the rumen. B2 proteins contain most of the albumins and glutelins. Ruminal degradation is 5 to 15 %/h. B3 proteins are prolamins, extensin proteins (cell wall associated proteins) and heat denatured proteins that did not undergo the Maillard reaction. They are degraded in the rumen at 0.1 to 1.5 %/h. Fraction C proteins consist of Maillard reaction proteins (heat damaged protein) and nitrogen bound to lignin. They are not degraded in the rumen.
20 Calculations Log of % nutrient remaining Protein Fraction A, B1 B2 B3 CHoursCalculate slope (change per hour) of each line.Slope = kd, has units of % of pool remaining that is lost per hour.
21 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.Protein requirements of the young growing ruminants and productive dairy cows often exceed the ruminal synthesis of microbial protein. Recognition of this requirement has resulted in the development of protein systems for beef and dairy cattle that distinguishes between dietary protein that is degraded in the rumen and dietary protein that escapes ruminal degradation.
22 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.
23 Protein content of common feedstuffs CP DIP UIP SolP NPN ADFIPFeedstuff %DM %CP %CP %CP %SolP %CPAlfalfa silageBarley silageCorn silageAlfalfa hayTimothy hayBarley strawBarley grainFermented feeds such as silages contain large amounts of soluble protein all of which is NPN. Hay proteins contain less soluble protein, but like the silages the protein is largely degradable in the rumen. Barley straw contains very little protein, much of which is lignified N and unavailable to the animal. Barley grain contains less soluble protein but the majority of the insoluble protein is readily degraded in the rumen. In feeds the unavailable N ranges from 3 to 15% of total N.
24 Protein content of protein supplements CP DIP UIP SolP NPN ADFIPPlant sources %DM %CP %CP %CP %SolP %CPCanola mealSoybean mealSoypass*Brewer’s grainsCorn distiller’s grCorn gluten meal*Commercial product: LignoTech USA, Inc.Protein supplements are defined as having a crude protein content of> 20%.Principle protein supplements of plant origin, such as canola meal and soybean meal, are derived from the meals after the oil has been extracted from the seeds. The majority of the protein in meals is degraded in the rumen. The degradablility of the protein in meals may be altered with the application of heat. Heat denatures the protein changing its solubility. SoyPass is a heat treated soybean meal with higher UIP than untreated soybean meal. The intestinal digestibility of the UIP is similar to untreated SBM. Increasing the UIP increases the bypass value of the protein.Distillers grains and brewers grains are by-products of the distilleries and breweries. Corn gluten meal is a by-product of corn starch and corn syrup manufacture, it is the residue remaining after removal of the larger part of starch and germ, and separation of the bran. All are high in bypass protein.
25 Protein content of protein supplements CP DIP UIP SolP NPN ADFIP%DM %CP %CP %CP %SolP %CPAnimal sourcesBlood mealFeather mealFishmealMeat and boneNon-protein nitrogen sourcesUreaAll animal protein sources are high in crude protein with high bypass value. There are limits to the amount of these protein sources that can be included in the ration because they tend to be less palatable. In 1997, the USDA prohibited the feeding of ruminant products to ruminant animals and thus there is little feeding of meat and bone meal. This move was in response to BSE.
26 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 intestineMethods to decrease the rate and extent of ruminal degradation involved the use of heat, chemical agents, or combination of both
27 Heat ProcessingHeat 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
28 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
29 Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteinsHeating is a common method of increasing the protein escape from the rumen. The mechanism of protection with heat treatment involves principally the Maillard reaction sequence between the -amino group of lysine residues and carbonyl compounds, usually reducing sugars such as glucose and fructose. The formation of the Shiff’s base is reversible but the modified Amadori compound and the polymers are nutritionally unavailable.
30 Heat ProcessingCareful control of heating conditions is required to optimize the content of digestible RUP.Under heating results in only small increasein digestible RUP.. Over heating reduces the intestinal digestibility of RUP through the formation of indigestible Millard products and protein complexes.
32 Chemical TreatmentChemical 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).
34 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.
36 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.
37 Characterization of Protein Sources Common protein supplements that are high in RUP are:Fish mealMeat 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)
47 Nitrogen transactions in the rumen Sources of nitrogen in the rumenDietary crude protein (true protein and NPN).Recycled microbial protein (bacteria and protozoa).Endogenous N (urea, abraded epithelial cells, salivary proteins).Crude protein (soluble and insoluble) is useful to describe the diet but when it comes to describing protein metabolism in the rumen, N transactions, which include all forms of N, must be considered.Factors affecting the extent of ruminal degradation of fermentable particulate proteins will depend on the structural characteristics of proteins, the number of hydrolysable sites, time of residence in the rumen, rate of solubilization, enzymatic concentration and pH.All interconversions of N in the rumen occur by the action of microorganisms.
48 Degradation of nitrogenous compounds by ruminal microorganisms Bacteria30 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.
49 Bacteria cont’d Protozoa Fungi R. amylophilus is the most active proteolytic bacteria. Important on starch-based diets.Breakdown of both soluble and insoluble protein in the rumen.ProtozoaMinor 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.FungiMinor role in protein degradation.
50 PROTEIN OLIGOPEPTIDES DIPEPTIDES AMINO ACIDS AMMONIA Dipeptidyl D. ruminantium, B. fibrisolvens, E. caudatumClostridium spp, E. simplex, E. budayiE. caudatum ecaudatum, E. ruminantium, E. maggiiFusobacterium spp., E. mediumL. multipara O. caudatus, P. ruminicolaP. multivesiculatum, R. amylophilus, S. ruminantiumO. joyonii, N. frontalis, S. bovis, P. communisOLIGOPEPTIDESDipeptidylpeptidaseS. bovis, R. amylophilus, P. ruminicolaDIPEPTIDESD. ruminantium, E. caudatumF. succinogenes, M. elsdenii, P. ruminicolaIsotricha spp., L. multipara, S. ruminantiumDipeptidaseProtein degradation in the rumenHydrolysis of protein by rumen microorganisms releases oligopeptides (4 to 9 AA), which are broken down to smaller peptides and finally to amino acids. Peptide breakdown in the rumen is a two-step process, dipeptidyl peptidases releases dipeptides (and tripeptides) from oligopeptides, followed by dipeptidases which cleave the resulting dipeptides into amino acids. The only common bacterial species that possesses high dipeptidyl peptidase is P. ruminicola. Ciliate protozoa also have high peptidase activity. Amino acid degradation is rapid yielding carbon skeletons and ammonia.AMINO ACIDSC. aminophilum, C. sticklandiiP. anerobius, B. fibrisolvens, P. ruminicolaM. elsdenii, S. ruminantium, E. caudatumIsotricha spp.AMMONIA
51 Properties of ammonia producing bacteria High NumbersLow ActivityButyrivibrio fibrisolvensMegasphaera elsdeniiPrevotella ruminicolaSelenomonas ruminantiumStreptococcus bovis> 109 per ml10 to 20 nmol NH3 min-1(mg protein)Low NumbersHigh ActivityClostridium aminophilumClostridium sticklandiiPeptostreptococcus anaerobius107 per ml300 nmol NH3 min-1(mg protein)Bacteria with amino acid deaminating capability fall into two groups: numerically abundant bacteria with low deaminiative activity, and few species with exceptionally high deaminative activity. The bacteria with high activity are atypical in that they ferment amino acids as their major source of energy and N. Ciliate protozoa also play a significant role in deamination of amino acids.
52 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 inthe rumenAmmonia
53 Influence of diet on proteolysis ConcentrateIncrease 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 forageIncrease in the proportion of proteolytic bacteria relative to total microbial population.Increased proteolytic activity is associated with feeding high proportions of cereals in the diet and fresh forage. Proteolytic activity of the rumen fluid, the numbers of proteolytic bacteria, and the predominant proteolytic species are all diet dependent but are related more to energy level and fermentability rather than level and type of protein.
54 Microbial protein synthesis in the rumen End products of proteolysis (ammonia, peptides, amino acids) are sources of N for the synthesis of microbial protein. Maximization of microbial protein synthesis requires a continuous supply of fermentable carbohydrates, ammonia, peptides, amino acids and other nutrients. A shortage of any of these can limit protein synthesis.
55 Factors Influencing Microbial Protein Synthesis AmmoniaMost 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 systemglutamate synthetase - glutamate synthase (GS-GOGAT)lower affinity, higher Km systemNADP-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).The high affinity enzyme system of ammonia assimilation is found under conditions of low ammonia concentration but is not significant under conditions of high ammonia concentration. The rumen concentrations in vivo would seldom be low.
56 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.
57 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 RatesIncreasing 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.As the rate of passage from the rumen increases there is greater opportunity for bacteria to pass out of the rumen.
58 Efficiency of Microbial Growth 1412108BCP/100 gm TDN6pHRate of passage425570Diet % of TDN (DOM)
59 Effect of dilution rate on YATP. This figure indicates that with increasing dilution rates a greater proportion of the energy derived from glucose is being used for net culture growth.Effect of dilution rate on YATP.
60 Reduces the amount of intraruminal N recycling (microbial protein turnover). This figure shows the relationship between dilution rate from the rumen and magnitude of microbial turnover at three different lysis rates: 0.1 (), 0.01 () and 0.001().
61 Intraruminal nitrogen recycling Turnover of bacteria and protozoa.30 to 55% of bacterial N75 to 90% of protozoal NCauses of microbial N recyclingEngulfment and subsequent digestion of bacterial cells by protozoaLysis due to autolytic enzymes, bacteriocins, or other soluble compounds in response to nutrient deprivation or interspecies competitionActivity of bacteriophages and mycoplasmas.It is generally accepted that protozoal predation has the greatest impact on bacterial N recycling. However, even in the absence of protozoa, a considerable amount of N is recycled between the rumen ammonia pool and microbes both indicating that many of the microorganisms secrete N or die and are fermented in the rumen.
62 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.One of the principle disadvantages of using urea as a source of NPN is that it is broken down too rapidly, resulting in ammonia overflow and inefficient N retention.
69 Urea recyclingBlood 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 activityDue to the location of the ureolytic microorganisms on the rumen epithelium these microorganisms are important in the hydrolysis of urea transferred across the rumen wall. These microorganisms move into rumen fluid when the epithelial cells they are attached to move into the rumen contents and are largely responsible for the urease activity in rumen fluid. The organisms in the fluid are important in hydrolyzing dietary and salivary urea.At low ruminal ammonia concentrations, there is an influx of urea to the rumen to maintain optimum levels of ammonia. Consequently, although N supply by the diet may be subject to great variation over a diurnal period, the diffusion process acts to stabilize ammonia concentrations in the rumen.
70 Factors involved in increasing the permeability of the rumen wall to urea The rate of diffusion of urea from the blood to the rumen is increased by increasing the level of nutrition. Increasing amounts of VFA promote proliferation of the epithelium lining the rumen wall and stimulate cell proliferation.
71 Composition of microbial protein reaching the intestine Hay Hay and Concsheep sheep sheep sheep2N in rumen digesta, gFungiProtozoaBacteriaN in rumen digesta, % of total microbial NFungiProtozoaBacteriaN in duodenal digesta, gFungiProtozoaBacteriaN in duodenal digesta, % of total microbial N flowFungiProtozoaBacteriaThe majority of microbial protein flowing from the rumen to the intestine is of bacterial origin. Bacterial protein contibutes from 70 to 95% of the microbial protein, protozoa from 5 to 30% and fungi less than 5%.
72 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.The undegraded fraction of dietary protein is of considerable interest because it can be manipulated by dietary treatment (heat) and by the use of specialized ingredients high in UIP.
73 Protein digestion in the abomasum HClProteinDenatured proteindisruption of non-covalent bondsuncoiling of proteinHClPepsinogen(inactive)Pepsin(hydrolysis bonds atcarboxylic end ofaromatic AA and Leu)The abomasum functions like the acid-secreting stomach in monogastric animals and humans. Hydrochloric acid (HCl) denatures the protein by disrupting the non-covalent bonds causing the protein to unfold. This is important for increasing the access of various proteases (enzymes that hydrolyze protein) to peptide bonds. Proteases are secreted in an inactive form (zymogen or proenzyme). HCl activates the proenzyme pepsinogen to pepsin by cleavage of a small peptide. Pepsin hydrolyzses peptide bonds at the carboxylic end of aromatic amino acids and Leu to yield smaller polypeptides.PepsinSmall polypeptidesfew amino acidDenaturedproteinpH 1.6 to 3.2
74 Secretion and activation of pancreatic and intestinal proteolytic enzymes PolypeptidesShort peptidesAAIntestinalendocrine cellCCK and SecretinCholecystokinin (CCK)Pancreaticacinar cellIntestinalmucosal cellEnterokinasePolypeptides, short peptides and amino acids signal to the intestinal endocrine cell to secrete into the blood the hormones cholecystokinin (CCK) and secretin. These hormones signal the pancreatic and intestinal cells to secrete proteases into the lumen of the intestinal tract. The proteases are secreted in an inactive form and require hydrolysis of a small peptide to become active.TrypsinogenTrypsinChymotrypsinogenProelastaseProcarboxypeptidaseChymotrypsinElastaseCarboxypeptidase
75 Sites of hydrolysis of proteolytic enzymes PancreasTrypsin Dibasic AA (Arg, Lys),C-terminal endChymotrypsin Aromatic C terminal peptidesElastase Neutral C terminal peptidesCarboxypeptidase C-terminal endIntestineEnteropeptidase N-terminal end
76 Digestion in the small intestine Pancreatic and intestinalproteasesAmino acidsDipeptidesTripeptidesPolypeptidesOligopeptidesIntestinal di- and tripeptidases(cell membrane and cytosol)DipeptidesTripeptidesAminoacids
77 Protein absorption Small intestine Major site of absorption Amino acids absorbed in the ileumDipeptides and tripeptides absorbed in the jejunumActive transport (energy dependent)9 carrier systems for amino acidsspecific for certain amino acids
78 Protein metabolism Intestinal cell Liver Glu, Asp, Gln metabolized by intestinal cellprovides 40% of energy requirementsLiverprotein synthesissynthesis of non-essential amino acidsC-skeletons catabolized for energy and the amine group metabolized to urea
79 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.
80 Routes of nitrogen excretion Urine (urea)Endogenous urinary N from the catabolism of tissue proteinsAbsorption and metabolism of excess ruminal ammonia.Catabolism of excess absorbed amino acidsFecesMicrobial N synthesized in and passed from the large intestine.Sloughed cells and secretions of the GI tract.Undigested unabsorbed dietary protein.
81 Feed Rumen Small intestine Large intestine Undegradable in rumen NPNIndigestibleDigestiblePeptidesaminoacidsPlasma ureaNH3RumenEnergyMicrobial NTissuesMaintenanceGrowthConceptusLactationWoolNH3PeptidesaminoacidsSmall intestineEndog NEnergyNH3Large intestineMicrobial NEndog NUrineFeces
82 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.
87 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.Because protozoa have such an impact on the amount of bacterial nitrogen recycled in the rumen, thereby reducing the amount of microbial N flowing from the rumen, there is a great interest in reducing or eliminating protozoa.
88 Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteinsHeating is a common method of increasing the protein escape from the rumen. The mechanism of protection with heat treatment involves principally the Maillard reaction sequence between the -amino group of lysine residues and carbonyl compounds, usually reducing sugars such as glucose and fructose. The formation of the Shiff’s base is reversible but the modified Amadori compound and the polymers are nutritionally unavailable.