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Hydroxy Methylthio Butyrate HMB (“N-Free” Met)

Hydroxy Methylthio Butyrate HMB (“N-Free” Met). 1. Ca Salt of HMB: a. Chandler et al. (1976): Increased Milk Fat Yield. b. Jones et al. (1988): 99% Degraded in the Rumen. c. Hosogai et al. (1989): Increased Blood Met. 2. Liquid Form of HMB (Alimet):

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Hydroxy Methylthio Butyrate HMB (“N-Free” Met)

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  1. Hydroxy Methylthio Butyrate HMB (“N-Free” Met) • 1. Ca Salt of HMB: • a. Chandler et al. (1976): Increased Milk Fat Yield. • b. Jones et al. (1988): 99% Degraded in the Rumen. • c. Hosogai et al. (1989): Increased Blood Met. • 2. Liquid Form of HMB (Alimet): • a. Stephenson et al. (1990): Basal Wool Growth Increased w/ Drench (~25% of Increase Obtained w/ Abomasal HMB). • b. Sklan & Tinksy (1996): RP-HMB Increased • Milk Yield on “Met-Limited” Diet. • c. Koenig et al. (1999): 50% Rumen Escaped of HMB. • d. McCollum et al. (2000): Direct Uptake in Stomach (?). • 3. Isopropyl-HMB: • a. Robert et al. (2001): 50% Escaped the Rumen. • b. St-Pierre & Sylvester (2005): Increased Milk, Fat & Protein. • c. Chen et al. (2009): HMBi Utilization ~= SmartAmine RPM. Ruminology-3--Utilization of Absorbed Protein

  2. Intestinal Protein Digestion & Amino Acid Utilization in the Ruminant Ruminology-3--Utilization of Absorbed Protein

  3. Intestinal Protein Digestion • The “Black Hole” of Ruminant Protein Nutrition. • Of Considerable Practical Importance. • Red Clover • Rumen-Protected Proteins • Measurement of Intestinal Protein Digestion. • Mobile Bag Technique • Pepsin-Pancreatin In Vitro Techniques • Dual-Cannulation of Intestine (Proximal Duodenum & Terminal Ileum) • Maintaining Dual Intestinally Cannulated Animals is Problematic. • “Good” Data are Badly Needed. Ruminology-3--Utilization of Absorbed Protein

  4. Utilization of Red Clover CP • NPN in Red Clover Silage Markedly Lower than Grass or Alfalfa Silages • Reduced Proteolysis in Silo due to Polyphenol Oxidase (PPO) Activity in Red Clover Ruminology-3--Utilization of Absorbed Protein

  5. Efficiency of N Utilization of RC and grass silages in the Rumen(Data from Finnish (MTT) omasal sampling studies) • Ruminal N-Efficiency of Red Clover Better than Grass Silages • Zero N balance at 168 g vs. 136 g CP/kg DM • Lower Ruminal Ammonia at Same Dietary CP Content • Reduced Protein Degradation • Improved Microbial Synthesis Ruminology-3--Utilization of Absorbed Protein

  6. Replacing Grass Silage with Red Clover Silage & NAN Flow and Protein Yield • Increased N Intake with Replacement of Grass by Red Clover Increased NAN Flow • But Increased Protein Flow Did Not Increase Milk Protein Yield • WHY? Ruminology-3--Utilization of Absorbed Protein

  7. Fecal CP higher for RC (MTT data) • Intercept of Lucas Test More Negative for Red Clover (RC) vs. Perenial Rye Grass (PG grass); • Fecal CP/kg DMI ~16 g Higher (RC) • “CP Digestibility Lower for RC” • (Pekka Huhtanen) • or • Impaired Utilization of Absorbed AA • (Glen Broderick) • PPO Depresses Protein Utilization as well as Rumen Degradation Ruminology-3--Utilization of Absorbed Protein

  8. Autoclaving Time on True Digestibility (Rat) & FDNB-Available Lys in Cottonseed Meal(Craig & Broderick, 1981) Ruminology-3--Utilization of Absorbed Protein

  9. Rat Growth as Function of Intake of FDNB-Lys or Truly Dig. Protein in Autoclaved CSM(Craig & Broderick, 1981) Filled = Autoclaved 30 to 60 min. Filled = Autoclaved 0 to 30 min. Ruminology-3--Utilization of Absorbed Protein

  10. Mobile Bag Technique In Situ Bags Inserted into Duodenum; Recovered at Terminal Ileum or in Feces. May Use Ruminal Pre-Incubations. Should Use an Acid-Pepsin Pre-Incubations. Nitrogen or AA Disappearance Designated as “Intestinal Digestibility”. Often Intestinal Digestibilities are > 95%. Recovering Mobile Bags in Feces May Confound Over-Estimates of Intestinal Digestibility. Ruminology-3--Utilization of Absorbed Protein

  11. Pepsin-Pancreatin In Vitro Method • Protein Exposed to Pepsin/HCl (pH 2). • pH Adjusted to 7.5. • Incubated with Mixed Pancreatic Enzymes. • Method very OLD: • Akeson & Stahmann (1964) • Craig & Broderick (1981) • Calsamiglia & Stern (1995) • In Vivo Proximal Duodenal to Terminal Ileal Digestion Remains a “Standard” (NOT = Availability). Ruminology-3--Utilization of Absorbed Protein

  12. Calsamiglia & Stern (1995) 3-Step In Vitro Method • In Situ Pre-Incubation Protein in the Rumen (16-h). • Mimic Escaped Protein (Assume Little Microbial Contamination) • Not a Measure of Protein Escape (also ftn of kd) • Protein Exposed to Pepsin/0.1 N HCl (1-h). • pH Adjusted to 7.6 & Incubated with Mixed Pancreatic Enzymes (24-h). 4. Has Shown Lower Digestibilities than Mobile Bag. Ruminology-3--Utilization of Absorbed Protein

  13. Effect of Method on Digestibility Estimated for Soybean Meals(Stern et al., 1997) • Mobile Bag Tech. 3-Step Technique • Phase Solv. SBM LS-SBM Solv. SBM LS-SBM • (CP Digestion, %) • Ruminal 73.1a 29.1b 80.6a 31.3b • Intestinal 99.3a 99.5a 77.6b 77.9b • Overall 99.8a 99.9a 95.9b 85.1c a-c(P < 0.05) Ruminology-3--Utilization of Absorbed Protein

  14. Digestible Lys vs. Bio-Available Lys & Lys Deposition in Growing Pigs(Rutherfurd et al., 1997) Ruminology-3--Utilization of Absorbed Protein

  15. Main References • Lapierre et al. 2005. The route of absorbed nitrogen into milk protein. Anim. Sci. 80:11-22. • Lobley. 2002. Protein turnover - What does it mean for animal production. CSAS Symp., 1-15. 3. J. P. Cant et al. 2002. Responses of the bovine mammary glands to absorptive supply of single amino acids. CSAS Symp., 27-43. Ruminology-3--Utilization of Absorbed Protein

  16. Protein Metabolism in the Cow’s Tissues Ruminology-3--Utilization of Absorbed Protein

  17. Whole Body N Metabolism in the Lactating Dairy Cow (Lapierre et al., 2005) Ruminology-3--Utilization of Absorbed Protein

  18. Amino Acid Metabolism in Tissues of the Lactating Dairy Cow(Lapierre et al., 2005) Ruminology-3--Utilization of Absorbed Protein

  19. EAA Uptake vs. Metabolism in the Lactating Cow • Amino acid His Leu Lys Met Phe Thr • Function • (g/day) • Absorbed 37 117 108 32 85 63 • Post-Liver 26 140 111 22 44 57 • Mammary • Uptake 23 101 88 24 43 40 • Milk Protein 22 82 68 23 42 37 • Milk/Post-Liver 0.82 a 0.58 0.61 1.01a 0.94a 0.64 (aNot different from 1.0) Ruminology-3--Utilization of Absorbed Protein

  20. EAA Absorption & Portal Appearance Blood vs. Presence in Body Protein (% of Ile, Leu, Lys, Phe, Thr + Val) Ruminology-3--Utilization of Absorbed Protein

  21. Ratio Appearance of EAA--Portal Drained Viscera (PDV) to Mesenteric Drained Viscera (MDV) a b bc c d e Ruminology-3--Utilization of Absorbed Protein

  22. Oxidation of EAA Across the Sheep GIT • Item Leucine Lysine Methionine Phenylalanine • GIT Oxidation 57.2a 1.1 7.6a -2.8 • (µmol/h) • % Whole Body 26.5a -1.0 9.9a -4.3 • Oxidation • % of Apparent 36.0a . . . 15.3a . . . • Uptake (aDifferent from 0) Ruminology-3--Utilization of Absorbed Protein

  23. Gut Amino Acid Metabolism • 1. Non-Essential Amino Acids (Glu & Gln--Major Energy Sources, Nucleic Acids Synthesis) • 2. Essential Amino Acids • a. Catabolism of Some EAA Related to Intake (Lys) • b. Essential Functions (e.g., Met--Methyl Groups) • c. Oxidative Losses of BCAA • 3. Endogenous Protein Losses • a. Digestive Enzymes & “Sloughing” (MFN ~ DMI) • b. Mucin Synthesis (High Thr Proteins) • c. Defensin Synthesis (Proteins High in Lys & Arg) Ruminology-3--Utilization of Absorbed Protein

  24. Liver Amino Acid Metabolism • 1. Major Site of Amino Acid Metabolism & GlcNG. • 2. Liver “Sees” Absorbed + Recirculated AA • a. Recirculated Amino Acids = 72-98%of Total • b. Relatively Constant Proportion of Each EAA (~12%) Extracted/Pass. • 3. Liver Not Active Regulator of Peripheral Supply. • a. Amino Acid Uptake for “Vital” Functions • b. Removal of Excess Amino Acids Ruminology-3--Utilization of Absorbed Protein

  25. Catabolism of Amino Acids • 1. Obligate Metabolic Functions (Gly -- Hippurate; • Met -- Methyl Groups) • 2. Losses Related to Increased Turnover (e.g., Incomplete Reutilization of Digestive Secretions) • Imbalance: Removal of Excess Amino Acids (Swine Production vs. Ruminant Production) 4. Utilization of AA for Gluconeogenesis (esp. Dairy Cow) Ruminology-3--Utilization of Absorbed Protein

  26. Is there a Constant Efficiency of Utilization of Metabolizable Protein & Amino Acids? Ruminology-3--Utilization of Absorbed Protein

  27. Plasma Lys Response to Abomasal Lys & Met Infusion in Growing Steers(Titgemeyer et al., JAS 71:421, 1988) 9.4% CP Steers = 383 kg BW; consumed 7.5 kg DM/d; gained 1.08 kg/d (Basal Supply of Digestible Lys = 53 g/d Duod. x 0.75 = 40 g Lys/d) Ruminology-3--Utilization of Absorbed Protein

  28. Plasma Lys Response to Abomasal Lys & Met Infusion in Growing Steers(Titgemeyer et al., 1988) (L-Lys + 9 g/d DL-Met) (L-Lys only) (DL-Met only) (Lys req. = 40 + 8 = 48 g/d) (Lys req. = 40 + 4 = 44 g/d) Ruminology-3--Utilization of Absorbed Protein

  29. Broken Stick vs. Logistic Model of EAA Utilization (Lapierre et al., 2005) Ruminology-3--Utilization of Absorbed Protein

  30. Relative Protein Efficiencies (Nout/Nin) • 1. 45-60% in Growing Pigs. • 2. Growing Cattle: • a. 20-30% in Young Cattle • b. < 10% at the Feedlot Stage. • 3. Lactating Cattle: • a. 30-35% at Peak Production. • b. 15-20% in Late Lactation. • c. 20-25% Over the Lactation Cycle. Ruminology-3--Utilization of Absorbed Protein

  31. Protein Turnover in the Animal Body • 1. Continual Synthesis & Breakdown of Body Protein. • 2. “Functions” of Protein Turnover: • a. Homeothermy--Basal Heat Production = 4-5 kcal/g Protein Synthesized (~30% for Protein TO) • b. Up/Down Enzyme Regulation • c. Tissue Remodeling • 3. Time Scale of Protein Turnover Varies. Ruminology-3--Utilization of Absorbed Protein

  32. Protein Intake vs. Turnover • Protein Body Protein • Animal Intake Synthesis Rate (g/day) Sheep 100 250 Human 75 300 Pig 250 500 Steer 1500 2500 Dairy Cow 3000 5000 Ruminology-3--Utilization of Absorbed Protein

  33. Protein Mass & Turnover 20% 15-20% 8-16% 50% 23-30% 15-20% Skin Muscle 3% 7-14% 35-45% Liver GIT 2% 25% 4-15% 5-7% 23% 32-45% Proportion of Whole Body: Tissue Protein Mass O2 Utilization Protein Synthesis Mammary Gland Ruminology-3--Utilization of Absorbed Protein

  34. Use of 15N15N-Urea to Quantify Urea Catabolism in GIT “Monomolecular Rxn” Infused: 15N15N-Urea + LiOBr Mass 30 N2 Gas Hydrolyzed: 14N15N-Urea + LiOBr Mass 29 N2 Gas Ruminology-3--Utilization of Absorbed Protein

  35. Urea Metabolism Quantified in Sheep Using 15N15N-Urea (Lobley et al., 2000) (BW = 50 kg; Intake =1000 g/d) Ruminology-3--Utilization of Absorbed Protein

  36. Urea Metabolism in Ruminants(Lapierre & Lobley, 2001) (average data from 15N15N-urea studies in cattle & sheep) Ruminology-3--Utilization of Absorbed Protein

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