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Protein Metabolism I ANS 520 Topics •Amino acid metabolism •Microbial protein contributions •Ruminal N digestion Protein Pathways Ruminant Protein Metabolism • Nitrogenous feed component, non-protein nitrogen components, endogenous – Ammonia for bacterial growth – Amino acids (AA) for animal needs (absorbed in small intestine) Feed Protein Acronyms NRC Publications Crude protein DIP (RDP) UIP (RUP) SolP, % CP NPN, % CP NDFIP, % CP ADFIP, % CP B1, B2, B3, % hr Total N x 6.25 Degraded intake protein Undegraded intake protein Soluble protein Nonprotein nitrogen Neutral detergent fiber insoluble protein Acid detergent fiber insoluble protein Rate constants for degradable fractions Protein • Analysis: Determine total N by Kjeldahl – All N NH4+ – Determine as NH3 – Total N x 6.25 = crude protein • Peptide bond: NH2 R1-C-C-NH O C-C=O R2 N-C-COOH H R3 Nitrogenous Compounds in Feeds • True proteins Polymers of AA (18 to 20 AA) linked by peptide bonds • Essential AA – Have to be present in the diet (absorbed) – Arg Lys Trp Leu Ile Val Met Thr Phy His » PVT TIM HALL • Nonessential amino acids (dispensable) – Synthesized in body tissues – Glu Gly Asp Pro Ala Ser Cys Tyr Proteins Peptides Amino acids Nitrogenous Compounds in Feeds • Nonprotein nitrogen – Nitrogen not associated with protein • Free amino acids, nucleic acids, amines, ammonia, nitrates, nitrites, urea • Crude protein – Total nitrogen x 6.25 – Proteins on average contain 16% nitrogen Protein Degradation in the Rumen Feed proteins Peptides Amino acids •Undegraded feed proteins •Escaped feed proteins •“Bypass proteins” Enzymes from protozoa and bacteria •Many species of bacteria involved •Bacterial enzymes are extracellular •Enzymes not in cell free rumen fluid •Both exopeptidase and endopeptidase activity Assumption in CNCPS: Enzymes (microorganisms) in excess – substrate limited Factors Affecting Ruminal Protein Degradation •Chemical nature of the proteins • Solubility – More soluble proteins degraded faster •Exceptions might include •egg ovalbumin, serum proteins • 3-dimensional structure – Affects solubility & availability • Chemical bonding •Disulfide bonds – Reduces degradation Factors Affecting Ruminal Protein Degradation • Physical barriers • Cell walls of plants • Cross linking of peptide chains – Reduces degradation • Aldehydes, Tannins • Feed intake • Rate of passage – Time proteins remain in the rumen • Feed processing • Rate of passage • Heat damage – Complexes with carbohydrates Estimating Ruminal Protein Degradation 1. In situ digestion Feed placed in Dacron bags suspended in the rumen Measure protein lost over time 2. Cannulated animals (rumen & duodenum) Measure protein flowing through duodenum Need to differentiate feed from microbes 3. In vitro incubation with rumen microbes Relative differences among proteins 4. In vitro digestion with fungal enzymes Protein Degradation In situ Log, % N remaining A - All degraded B - Partly degraded Slope = degradation rate C - Not degraded Digestion time, hr Protein Degradation DIP (RDP) = A + B[Kd/(Kd+Kp)] DIP = Degraded intake protein Kd = degradation rate, %/h Kp = passage rate, %/h UIP (RUP) = B[Kp/(Kd+Kp)] + C UIP = Undegraded intake protein Feed Protein Fractions (CNCPS & NRC) NPN Soluble Feed Insoluble - A Sol Proteins - B1 Insoluble - B2 Insoluble - B3 Indigestible - C Protein Fractions In Feeds Laboratory Analysis A - Soluble in buffer (borate-phosphate) and not precipitated by tungstic acid B1 - Soluble in buffer and precipitated by tungstic acid B2 - Insoluble in buffer = (Insol protein) - (protein insol in neutral detergent) B3 - Insoluble in buffer = (Insol in neutral detergent) - (Insol in acid detergent) C - Insoluble in buffer and acid detergent Kd Values for Feed Proteins Fraction A B1 B2 B3 C Kd, %/h Infinity 120 to 400 3 to 16 0.06 to 0.55 Not degraded Kp Values Wet forages Kp = 3.054 + 0.614X1 Dry forages Kp = 3.362 + 0.479X1 – 0.007X2 – 0.017X3 Concentrates Kp = 2.904 + 1.375X1 – 0.020X2 X1 = DMI, % Body Wt X2 = Concentrate, % of ration DM X3 = NDF of feedstuff, % DM “Bypass proteins” • Proteins that are not extensively degraded in the rumen • Natural •Corn proteins, blood proteins, feather meal •Modification of feed proteins to make them less degradable •Heat - Browning or Maillard reaction •Expeller SBM, Dried DGS, Blood meal •Chemical •Formaldehyde •Polyphenols •Tannins •Alcohol + heat •Usually some loss in availability of amino acids - lysine Average Ruminal Degradation of Several Proteins Used in Level 1 Soybean meal (Solvent processed) Soybean meal ( Expeller processed) Alfalfa Corn proteins Corn gluten meal Corn gluten feed Dried distillers grains Blood meal Feather meal Urea 75% 50% 80% 62% 42% 80% 55% 20% 30% 100% Degradation of NPN Compounds Activity associated with microorganisms • Urea CO2 + 2 NH3 High concentrations of urease activity in the rumen Low concentrations of urea in the rumen • Biuret 2 CO2 + 3 NH3 Low activity in the rumen • NO3 NH3 Fate of Free Amino Acids in the Rumen • Amino acids not absorbed from the rumen • Concentrations of free AA in the rumen very low • Amino acids and small peptides (up to 5 AA) transported into bacterial cells • Na pumped out of cells – Uses ATP • Na gradient facilitates transport of AA by a carrier Utilized for synthesis of microbial proteins Amino acids metabolized to provide energy • • Amino Acid Degradation in the Rumen Amino acids NH3 CO2 Keto acids VFA • Enzymes from microorganisms Intracellular enzymes • Peptides probably hydrolyzed to amino acids and then degraded • NH3, VFA and CO2 absorbed from rumen Amino Acid Fermentation Valine Leucine Isoleucine Isobutyrate Isovalerate 2-methybutyrate Alanine, glutamate, histidine, aspartate, glycine, serine, cystein and tryptophan pyruvate Threonine, homoserine, homocyseine and methionine Ketones Control of Amino Acid Fermentation When CHOH is ample for growth, incorporation of amino acids into protein is favored • Majority of transported amino acids and peptides do not go through ammonia pool When CHOH supply is limiting growth, amino acids are fermented for energy • There is an increase in amino acids going through the ammonia pool Amino Acid Fermenters in the Rumen High numbers Low activity Butrivibrio fibrisolvens Measphaera elsdenii Selenomonas ruminantium 109 per ml 10 to 20 NMol NH3 per min per mg protein Monensin resistant Involved in CHOH fermentation Low numbers High activity Clostridium aminophilum Clostridium sticklandii Peptostreptococuss anaerobius 107 per ml 300 NMol NH3 per min per mg protein Monensin sensitive Ferment CHOH slowly or not at all Microbial Protein Synthesis •End product of protein degradation is mostly NH3 •Protein synthesis •Fixation of N in organic form •Synthesis of amino acids •Synthesis of protein(s) Rumen microbes • Bacteria (50% CP) • Protozoa (20-60%, avg 40% CP) • Bacteria major player, % of microbial N entering SI from protozoa < 10% • N source for microbes – Diet protein – Non protein N – Recycled N Microbial N • Microbial N entering SI (% of non-ammonia N) – High protein diets -40% – Low protein diets -60% – Exclusive NPN diet -100% • Limiting factors would include C and/or energy source Nutritive Value of Microbial N • • • • Increases value of low quality feed N Decreases value of high quality feed N Animal can survive on non-protein N Can survive on low amounts of recycled N Bacterial Protein Synthesis in the Rumen NH3 Amino acids & Peptides VFA Amino acids Fermentation CHOH VFA Microbial protein synthesis related to: 1. Available NH3 and amino acids (DIP) 2. Fermentation of CHOH - Energy Microbial proteins Microbial Requirements Bacteria Nitrogen • Mixed cultures NH3 satisfies the N requirement Cross feeding can supply amino acids • Pure cultures Fiber digesters require NH3 Starch digesters require NH3 and amino acids Peptides can be taken up by cells Branched-chain fatty acids • Required by major rumen cellulolytic bacteria Energy from fermentation • Need energy for synthesis of macromolecules Role of Protozoa •Do not use NH3 directly •Engulf feed particles and bacteria • Digest proteins • Release amino acids and peptides into rumen • Use amino acids for protein synthesis • Protozoa engulf bacteria • Protozoa lyse easily – May contribute little microbial protein to the animal Efficiency of Microbial Growth Grams microbial N/100 g organic matter digested Ranges from 1.1 to 5.0 1. Kind of diet Forages > Grain 2. Level of feeding High > Low 3. Rate of passage Fast > Slow 4. Turnover of microbial cells Younger cells turnover less than aging cells 5. Maintenance requirement of cells Microbes use energy to maintain cellular integrity 6. Energy spilling Dissipation of energy different from maintenance Most apparent when energy is in excess Efficiency of Microbial Growth Slow passage Low rumen pH Low quality forages slow passage Bacteria use energy to pump protons TDN, % feed DM Microbial Growth in The Rumen Nutrients available to microbes 1. DIP - NH3, peptides, amino acids • CNCPS adjusts for inadequate available N 2. Energy from the fermentation • • Growth rate related to Kd of CHOH Quantity of cells related to CHOH digested CNCPS assumes microbes digesting non-fiber and fiber CHOH both have a maximum yield of 50g cells/100g CHOH fermented 3. Other - branched-chain acids, minerals