1. Protein Metabolism I ANS 520

2. Topics Amino acid metabolism Microbial protein contributions Ruminal N digestion

4. Protein Pathways

5. 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)

6. Feed Protein Acronyms NRC Publications Crude proteinTotal N x 6.25 DIP (RDP)Degraded intake protein UIP (RUP)Undegraded intake protein SolP, % CPSoluble protein NPN, % CPNonprotein nitrogen NDFIP, % CPNeutral detergent fiber insoluble protein ADFIP, % CPAcid detergent fiber insoluble protein B1, B2, B3, % hrRate constants for degradable fractions

7. Protein Analysis: Determine total N by Kjeldahl –All NNH 4 + –Determine as NH 3 –Total N x 6.25 = crude protein Peptide bond: NH 2 R 1 -C-C-NH OC-C=O R 2 N-C-COOH H R 3

8. 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

9. 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

10. Protein Degradation in the Rumen Feed proteinsPeptides 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

11. 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

12. 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

13. 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

14. Protein Degradation In situ A - All degraded B - Partly degraded Slope = degradation rate C - Not degraded Digestion time, hr Log, % N remaining

15. 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

16. Feed Protein Fractions (CNCPS & NRC) Soluble Insoluble NPN - A Sol Proteins - B1 Insoluble - B2 Insoluble - B3 Indigestible - C Feed

17. 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

18. Kd Values for Feed Proteins FractionKd, %/h AInfinity B1120 to 400 B23 to 16 B30.06 to 0.55 CNot degraded

19. 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

20. “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

21. Average Ruminal Degradation of Several Proteins Used in Level 1 Soybean meal (Solvent processed)75% Soybean meal ( Expeller processed)50% Alfalfa80% Corn proteins62% Corn gluten meal42% Corn gluten feed80% Dried distillers grains55% Blood meal20% Feather meal30% Urea 100%

22. Degradation of NPN Compounds Activity associated with microorganisms UreaCO 2 + 2 NH 3 High concentrations of urease activity in the rumen Low concentrations of urea in the rumen Biuret2 CO 2 + 3 NH 3 Low activity in the rumen NO 3 NH 3

23. 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

24. Amino Acid Degradation in the Rumen NH 3 CO 2 Amino acidsKeto acidsVFA Enzymes from microorganisms Intracellular enzymes Peptides probably hydrolyzed to amino acids and then degraded NH 3, VFA and CO 2 absorbed from rumen

25. Amino Acid Fermentation ValineIsobutyrate LeucineIsovalerate Isoleucine2-methybutyrate Alanine, glutamate, histidine, aspartate, glycine, serine, cystein and tryptophanpyruvate Threonine, homoserine, homocyseine and methionineKetones

26. 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

27. Amino Acid Fermenters in the Rumen High numbersLow numbers Low activityHigh activity Butrivibrio fibrisolvens Clostridium aminophilum Measphaera elsdenii Clostridium sticklandii Selenomonas ruminantium Peptostreptococuss anaerobius 10 9 per ml 10 7 per ml 10 to 20 NMol NH 3 300 NMol NH 3 per min per min per mg protein per mg protein Monensin resistant Monensin sensitive Involved in CHOH Ferment CHOH slowly or fermentation not at all

28. Microbial Protein Synthesis End product of protein degradation is mostly NH 3 Protein synthesis Fixation of N in organic form Synthesis of amino acids Synthesis of protein(s)

29. 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

30. 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

31. 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

32. Bacterial Protein Synthesis in the Rumen NH 3 Amino acids & Peptides VFA Amino acidsMicrobial Fermentationproteins CHOH VFA Microbial protein synthesis related to: 1. Available NH 3 and amino acids (DIP) 2. Fermentation of CHOH - Energy

33. Microbial Requirements Bacteria Nitrogen Mixed cultures NH 3 satisfies the N requirement Cross feeding can supply amino acids Pure cultures Fiber digesters require NH 3 Starch digesters require NH 3 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

34. Role of Protozoa Do not use NH 3 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

35. Efficiency of Microbial Growth Grams microbial N/100 g organic matter digested Ranges from 1.1 to 5.0 1. Kind of dietForages > Grain 2. Level of feedingHigh > Low 3. Rate of passageFast > 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

36. Efficiency of Microbial Growth TDN, % feed DM G BCP/100 g TDN 813 Slow Low rumen passagepH Bacteria Low quality use energy to forages slow pump protons passage

37. Microbial Growth in The Rumen Nutrients available to microbes 1.DIP - NH 3, 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