The Amino Acid Content of Rumen Microbes, Feed, Milk and Tissue after Multiple Hydrolysis Times and Implications for the CNCPS M. E. Van Amburgh, A. F. Ortega, S. W. Fessenden, D. A. Ross, and P. A. LaPierre Dept. of Animal Science Outline How accurate do we need to be with AA predictions? Model guided research approach What are the current approaches to AA analysis? What are other approaches to AA analysis? Microbial, feed, milk and tissue AA data from longer term hydrolysis CNCPS evaluation of microbial data Summary Measuring AA flow in a cow How much AA are we talking about? Real data (omasal flow study) Cows consuming 2 kg dry matter, making 41 kg of milk per day ~2,3 g/d of AA flowing to small intestine 1 g Lys (6.7% of flow) 6 g Met (2.6% of flow) How precise do we need to predict supply? 1 g? g? 1 g?! 1 g is 1/2,3 of total AA flow, or.43% We supplement AA at 2 4 g/d 3/23 = 1.3% Fessenden, 216 Modeling to Predict AA Balance Four Pieces To The Nitrogen/AA Part of the Puzzle Total amino acid requirements Nitrogen components at the duodenum What is most limiting? Digestibility in the small intestine Amino acid profile of each component
Model Guided Research: CNCPS v7 Non Ammonia N Predicted vs Observed and Predicted vs Observed Lysine all on an N basis Possible Sources of Error/Variation 8 3 Missing information.. Observed non-ammonia N (g/d) 7 6 4 3 2 1 Observed Lys (g/d) 2 2 1 1 Microbial AA are not represented correctly Endogenous AA are not represented correctly Feed AA are not represented correctly 1 2 3 4 6 7 8-1 Predicted non-ammonia N (g/d) 1 1 2 2 3 Predicted Lys (g/d) Amino Acid Determination Amino acid content of feeds has historically been determined by single time point hydrolysis, as this represents a compromise between maximal release of AA from the matrix while minimizing the loss of acid labile AA (Rutherfurd, 29) New data suggests that determination of AA at multiple time points (2, 4, 6, 12, 18, 21, 24, 3, 48, 72, 12 and 168 h) followed by least squares non linear regression provides more accurate estimates of the AA profile (Darragh and Moughan, 2) This approach has been utilized in purified protein (Darragh et al., 1996), milk protein (Rutherfurd et al., 28) and common animal feedstuffs (Rutherfurd, 29) Previous work in our laboratory indicated that to obtain the greatest release of branched chain AA in forages, hydrolysis times needed to be greater than 21 hr; while Ile release was greatest at 7 hr (Ross, 24) Debbie Ross, M.S. Thesis, 24
Debbie Ross, M.S. Thesis, 24 Debbie Ross, M.S. Thesis, 24 Data Generated to Evaluate AA Methods and AA Content Microbial samples from 2 experiments published Fessenden et al. 217 Feed from 3 lactating cattle experiments Bulk tank milk from CURC over 4 days Tissue from 2 heifer slaughter balance experiments (Diaz and Meyer) Amino Acid Methods Amino acid content of all samples was determined by HPLC following hydrolysis for 21 h or 24 h at 11 C in a block heater (Gehrke et al., 198) Microbes, feeds, milk and bovine tissues were also hydrolyzed for 2, 4, 6, 12, 18, 21, 24, 3, 48, 72, 12 and 168 h to evaluate the rate of release of each AA. Time points chosen based on similar analysis performed on milk proteins (Rutherfurd et al., 28). The entire time course was performed twice for each sample, and the reported values are the mean of the 2 determinations.
Sulfur Amino Acid Analysis Methods For the sulfur containing AA, Met and Cys, samples containing 2 mg of N and the internal standard, norleucine, were pre oxidized with 1 ml performic acid to be analyzed as cysteic acid and methionine sulfone. Same time points Microbial AA profile and all other profiles Multiple time point hydrolysis 2, 4, 6, 12, 18, 21, 24, 3, 48, 72, 12, 168 hrs Least squares non linear regression 3 2 2 1 1 Lys 24 48 72 96 12 144 168 2 1 1 Met 24 48 72 96 12 144 168 3 2 2 1 1 Val 24 48 72 96 12 144 168 1 Effect of hydrolysis time (h) on content of essential AA (mg/g DM) from freeze dried isolations of omasal bacteria ( ) and protozoa ( ) using least squared regression for each dataset. 2 1 1 2 1 1 Arg Bacteria Protozoa 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Ile Bacteria Protozoa 12 24 36 48 6 72 84 96 18 12 132 144 16 168 1 8 6 4 2 3 2 2 1 1 His Bacteria Protozoa 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Leu 12 24 36 48 6 72 84 96 18 12 132 144 16 168
Effect of hydrolysis time (h) on content of essential AA (mg/g DM) from freeze dried isolations of omasal bacteria ( ) and protozoa ( ) using least squared regression for each dataset. 3 2 2 1 1 2 2 1 1 2 2 1 1 Lys 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Phe 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Trp 12 24 36 48 6 72 84 96 18 12 132 144 16 168 2 1 1 2 2 1 1 3 2 2 1 1 Met 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Thr 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Val 12 24 36 48 6 72 84 96 18 12 132 144 16 168 Comparison of the AA composition (g/1 g AA) of omasal bacteria determined using multiple hydrolysis time point or single hydrolysis time point methods Method 9 % CI AA Single Multiple S M SED 2 Lower Upper EQ 3 Arg. 4.73.27.3.6.48 No His 2.12 2.11.1.14.8.86 No Ile 4. 4.62.8.46 3.46 2.31 No Leu.6.32.28.26 1.3 1.91 No Lys 7.4 7.17.37.4.11.63 No Met 4.49 4.63.14.36 2.41 2.13 No Phe 6..77.23.9.31.77 No Thr.49.3.4.1.69.6 No Trp.97.77.2.3.1.41 No 18 Val.92 6.32.41.32 2.41 1.6 No Comparison of the AA composition (g/1 g AA) of omasal protozoa determined using multiple vs. single time point hydrolysis methods Method 9 % CI AA Single Multiple S M SED 2 Lower Upper EQ 3 Arg.3.26.9.1.84 1.3 Yes His 2.3 2.2.1.1.3. Yes Ile 3.8 4.39.9.6.94.24 Yes Leu 6.11 6.2.14.41 2.7 2.42 No Lys 8.81 8..26.6.1.62 Yes Met 3.14 3.77.62.47 3.8 2.34 No Phe 6.49 6.8.8.24 1.61 1.4 No Thr.41.34.7.3.13.26 Yes Trp 4.76 4.9.19.27 1.9 1.2 No 19 Val 4.6 4.7.1.4.38.18 Yes Equivalency chart for difference in AA composition (g/1 g AA) of bacteria determined using multiple vs. single time point hydrolysis methods. Open circles ( ) represent the mean difference and error bars represent the 9% confidence interval around the mean difference. Shaded region represents equivalency defined as.4 to.4 g/1 g bacterial AA. difference, g/1g AA. 4. 3. 2. 1.. -1. -2. -3. -4. -. Essential AA Non-essential AA Arg His Ile Leu Lys Met Phe Thr Trp Val Ala Asp Cys Glu Gly Pro Ser Tyr 2
Equivalency chart for difference in AA composition (g/1 g AA) of protozoa difference, g/1g AA. Essential AA Non-essential AA 4. 3. 2. 1.. -1. Forages and Feeds Feeds from omasal flow studies and other AA related studies were hydrolyzed for 2, 4, 6, 12, 18, 21, 24, 3, 48, 72, 12 and 168 h to evaluate the rate of release of each AA and compared to 24 hr hydrolysis. -2. -3. -4. -. Arg His Ile Leu Lys Met Phe Trp Thr Val Ala Asp Cys Glu Gly Pro Ser Tyr 21 Effect of hydrolysis time on release of Essential AA (mg/g DM) from two concentrates and two forages Effect of hydrolysis time (h) on release of lysine (mg/g DM) from two concentrates and two forages. LYS ALFALFA LYS GROUND CORN GRAIN AA, MG/G DM 4. 4 3. 3 2. 2 1. 1. 2 4 6 8 1 12 14 16 18 HYDROLYSIS TIME (H) AA, MG/G DM 1.6 1.4 1.2 1.8.6.4.2 2 4 6 8 1 12 14 16 18 HYDROLYSIS TIME (H) 23 AA, MG/G DM LYS CORN SILAGE.8.7.6..4.3.2.1 2 4 6 8 1 12 14 16 18 HYDROLYSIS TIME (H) AA, MG/G DM LYS SOYBEAN MEAL 16 14 12 1 8 6 4 2 2 4 6 8 1 12 14 16 18 HYDROLYSIS TIME (H)
The amino acid composition (mg/g DM) of carcass tissues from Diaz et al., (21) and Meyer (2) analyzed at 21 and 168 hr of hydrolysis and calculated by logistic regression of the content of the residues. 21h 168h 21 h 168 h Arg 13.6 1.6 2. His 8.7 8..7 Ile 8. 1.1 2.1 Leu 16.6 17.1. Lys 1.6 12.2 3. Phe 1.2 1.6.4 Thr 7. 7.. Val 8.4 1.1 1.7 Met 8. 8.3.2 2 The average amino acid composition (mg/g DM) of four milk samples from CURC over four days analyzed at 21 and 168 hr of hydrolysis 21 h 168 h 21 h 168 h Arg 4.33.1.68 His 4.83 2.97 1.86 Ile 4.3 6.4 1.1 Leu 1.1 11.12.97 Lys 9.9 7.83 1.26 Phe 6.8 7.48.89 Thr 4.37 4.93.6 Val.99 7.46 1.47 Met.3.6.24 26 Model evaluation Model evaluation Lysine Using updated AA profile of microbial protein Literature dataset: Omasal studies (n=16) with 61 treatment means Same dataset used to evaluate v6. and v7. Predicted N and AA flows were compared with reported values Random effect of study included in mixed model analysis 27 Observed Lys (g/d) Original v.7 Updated v.7 3 2 2 1 1 1 1 2 2 3 Predicted Lys (g/d) Observed Lys (g/d) 3 2 2 1 1 1 1 2 2 3 Predicted Lys (g/d) Var. Comp. MSPE Part. (%) AA R 2 BLUP R 2 MP RMSE Slope Int. Study Res. CCC RMSPE U m U s U r Lys old.92.8 1.69 8 78 21.36 61 8 9 11 Lys new.94.6 2.79 27 81 19.76 23 6 21 73 28
Model evaluation Methionine Original v.7 Updated v.7 1 1 1 Model evaluation Histidine Original v.7 Updated v.7 1 8 8 8 8 Observed Met (g/d) 6 4 2 2 2 4 6 8 1 Predicted Met (g/d) Observed Met (g/d) 6 4 2 2 2 4 6 8 1 Predicted Met (g/d) Var. Comp. MSPE Part. (%) Observed His (g/d) 6 4 2 2 4 6 8 1 2 Predicted His (g/d) Observed His (g/d) 6 4 2 2 4 6 8 1 2 Predicted His (g/d) Var. Comp. MSPE Part. (%) AA R 2 BLUP R 2 MP RMSE Slope Int. Study Res. CCC RMSPE U m U s U r Met old.94.42 3.67.78 9 88 12.6 12 2 6 74 Met new.9.43 1.67 8 89 11.4 21 66 11 23 29 AA R 2 BLUP R 2 MP RMSE Slope Int. Study Res. CCC RMSPE U m U s U r His old.91.61 4.2.82.8 74.9 2.1.6 2.7 3 17 8 His new.9.6 7.74 1 7 3.71 9 8 34 8 3 Predicted Lys Requirements VS Lysine Supply Lysine Use Expressed Relative To Metabolizable Energy Or Metabolizable Protein 16 Model predicted Lys requirement (g/d) 14 12 1 8 6 4 Ratio of predicted Lys requirement:supply 1.2 1.1 1..9.8.7.6..4 R 2 =.7 RMSE =.6 Ratio of predicted Lys requirement:supply 1.2 1.1 1.9.8.7.6..4 2 1 1 2 2 3 3 Digested Lys (g/d) Higgs and Van Amburgh, 216.3.2 1. 2. 3. 4.. 6. Digestible Lys supply (g Lys/Mcal ME).3.2 4. 6. 8. 1. 12. 14. Digestible Lys supply (g Lys/1g MP) Higgs and Van Amburgh, 216
Optimum Supply Of Each EAA Relative To Metabolizable Energy AA R 2 from our Efficiency evaluation Lapierre et al. (27) g AA/ Mcal ME % EAA Arg.81.61.8 2.4 1.2% His.84.77.76.91 4.% Ile.74.67.67 2.16 1.8% Leu.81.73.61 3.42 17.% Lys.7.67.69 3.3 1.1% Met.79.7.66 1.14.7% Phe.7.8.7 2.1 1.7% Thr.7.9.66 2.14 1.7% Trp.71.6 N/A.9 2.9% Val.79.68.66 2.48 Lys and Met requirements 14.9%,.1% Schwab (1996) 12.4% Lys and Met requirements 14.7%,.3% Rulquin et al. (1993) Implications The AA methods used to date do not fully recover all AA from the matrix need longer term hydrolysis Longer hydrolyses destroy some AA so need to look for the maximum levels testing regression vs specific time points The use of 6M HCl is not efficient and this makes sense cattle (mammals) have enzymes in addition to acid/pepsin hydrolysis to extract nutrients in a short GIT transit time From a modeling perspective, both the supply side and the demand side needs to be re evaluated 34 Implications Microbial AA contents in the CNCPS are being updated and evaluated Feed AA content in the feed library are being evaluated This is difficult because we cannot analyze all the feeds in the library Looking for some patterns or changes in recovery by feed type, e.g corn silage, alfalfa, soy, canola, etc. Milk and tissue AA are also being updated with the new information Efficiencies of use of AA have to be re calculated Thanks for your attention 3 36